Polynucleotide intercalator interceptors and inhibitors

ABSTRACT

A method of using modified xanthine molecules as a binding agent is disclosed. Xanthine molecules with at least one substitution of a methyl group at the N1, N3, N7, or N9 position bind to intercalating molecules efficiently. This method can be applied to inhibiting intercalating molecules from binding to nucleic acids, as well as removing intercalating molecules that have been bound to nucleic acids. This method can also be applied to synthesize an efficient drug delivery system for compounds that have low solubility in aqueous media, including anti-neoplastic agents. The method can also be applied to flurosecently labeling nucleic acids.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provision Patent ApplicationNo. 60/251,906 filed Dec. 6, 2000, and entitled “Interaction of DNA withXanthines, Acridine Orange, and Caffeine,” which is expresslyincorporated herein by reference in its entirety.

RIGHTS IN THE INVENTION

This invention was made in part with United States Government supportunder a grant number DE00152 awarded by the National Institute of DentalResearch.

BACKGROUND OF THE INVENTION

This invention relates generally to the field of molecular biology andmore specifically to the field of treatment and prevention of DNAbinding agents.

Concern for the detrimental and carcinogenic interaction of drugs andchemicals with oral, respiratory tract, and other tissues continues toencourage the study of systems offering potential reduction of cancerrisk. One such approach is the interaction of caffeine and otherxanthine-like compounds with polynuclear aromatic hydrocarbons andrelated carcinogenic materials. This interaction likely occurs through anovel mechanism known as polarization bonding. Polarization bonding isthe interaction of two planar molecules via a sandwich-like stacking toform a stable complex. This plane-to-plane packing of alternatepolarizing and polarizable molecules results in a shortening of the vander Waals distances and thereby provides for the stability and thestrength of the complexes formed. Polynuclear aromatic hydrocarbons(PAH), a class of carcinogens found in smokeless tobacco and tobaccosmoke are proposed to be initiators of oral and respiratory tractcancer, respectively. FIG. 1-1 shows chemical structures of same PAHs.It should be noted that both xanthines and PAHs are planar molecules,and that planarity has been described as a requirement forcarcinogenicity of PAH's as well as for complexation. The phenomenon ofpolarization bonding, specifically in the formation of xanthine-PAHcomplexes, has been observed, but has seldom been studied from a cancerprevention point of view. The complexation of PAH's to deoxyribonucleicacid may also be due to polarization bonding with the purine bases ofDNA.

The effects of polarization bonding on carcinogenesis and mutagenesishas not specifically been reported in the literature based on Medlineand Cancerlit computer searches, however the interaction of polynucleararomatic hydrocarbons with caffeine and other xanthine-like compoundshas been reported. For example, the measurement of the solubility andoptical properties of PAHs in solutions of caffeine, TMU, or DNAindicate similar mechanisms in the formation of complexes. Thecomplexation of PAH's to deoxyribonucleic acid may be due specificallyto the purine bases comprising DNA. As noted the xanthines and PAHs areplanar molecules, and planarity has been described as a requirement forcarcinogenicity of PAH's (Pullman, 1955, 1964) as well as complexation(Jones and Neuworth, 1949; Leela and Mason, 1957; Pullman and Pullman,1958; DeSantis et al., 1960, 1961; Liquori et al., 1962; Van Duuren andBardi, 1963; Van Duuren, 1964).

Several laboratories have recently reported on the interaction ofxanthines and xanthine analogs with carcinogenic polyaromatic DNAintercalators (Larsen et al., 1996; Traganos et al., 1991a, 1991b, 1993;MacLeod et al., 1991,1993; Kapuscinsid and Kimmel, 1993). FIG. 1-2 showschemical structures of example xanthines. These same xanthines bind withDNA intercalators through an interaction referred to as polarizationbonding (McKeown et al., 1951). Polarization bonding, is the π-πinteraction and stacking of two planar molecules, where one ispolarizing (xanthine) and the other is polarizable (DNA intercalator).The binding of these carcinogens to certain xanthines has been shown toinhibit or reduce their binding to dsDNA (Booth and Boyland, 1953;Shoyab, 1979; Traganos et al., 1991a; MacLeod et al., 1991). Planarityhas been described as a necessary requirement for polarization bondingcomplexation (McKeown et al., 1951; Harding and Wallwork, 1953, 1955;Wallwork, 1961; Liquori et al., 1962; Miller, 1970; Huberman et al.,1976) as well as for intercalation into dsDNA (Booth and Boyland, 1953;DeSantis et al., 1960, 1961; Van Duuren, 1964; Pullman, 1964).Polarization bonding has been proposed as the mechanism for the bondingof carcinogenic polyaromatic hydrocarbons, such as benzo[a]pyrene anddimethyl benzanthracene to the purine bases adenine and guanine withinDNA as well as to other xanthine-like molecules (DeSantis et al, 1960,1961). The specific mechanism by which xanthine-like molecules inhibitpolyaromatic DNA intercalating agents (DNA-IAs) from intercalation intodsDNA has been suggested to be due to the formation of a polarizationbonding complex between the xanthine and the DNA-IA (Traganos et al.,1991a, 1991b; Kapuscinski and Kimmel, 1993; Tachino et al., 1994; Larsenet al., 1996). The degree of reversibility of the intercalation ofDNA-IAs into DNA, in vitro and in vivo, remains to be established.

SUMMARY OF THE INVENTION

According to one embodiment, the present invention is directed to amethod of inhibiting complexation of intercalating molecules withpolynucleotides. According to a preferred embodiment, the presentinvention is directed to a method comprising complexing a xanthinemolecule to an intercalating molecule. According to one implementation,xanthine is contacted with the intercalating molecule, wherein thexanthine molecule may comprise at least one methyl group at the N1, N3,N7, and/or N9 positions.

According to another embodiment of the present invention, the xanthinemolecule may also comprise at least one substitution of an oxygen groupor a chlorine group at the C8 position. The present invention may beused on various intercalating molecules, including but not limited tothe group consisting of acridine orange, doxorubicin, doxorubicinhydrochloride, novantrone, elipticine, ethidium bromide, Hoeschst 33258,aflatoxin and/or mixtures thereof.

The present inventive methods and/or compounds are also useful in theinhibition of intercalating molecules that are planar polyaromatichydrocarbon molecules., e.g., benzo(a)pyrene and dimethyl benzathracene.

According to another embodiment, porphyrins, instead of xanthines may beused to contact with the intercalating molecule.

According to still further embodiments, and depending upon theintercalator and/or intercalators, the number of methyl groupsubstitutions on the xanthine molecule is increased. For example, thexanthine molecule of the present invention may be selected from thegroup consisting of 1-methyl xanthine, 1,3-dimethyl xanthine,3,7-dimethyl xanthine, 1,7-dimethyl xanthine, 1,3,7-trimethyl xanthine,1,3-dimethyl xanthine, 1,3-dimethyl-8-oxy xanthine,1,3-dimethyl-8-chloro xanthine, 1,3,7,9-tetramethyl-8-oxy xanthine, andmixtures thereof.

According to a preferred embodiment of the present invention (anddepending upon the intercalator or intercalators) the binding affinitybetween the intercalating molecule and the xanthine molecule ispreferably at least 150 MD⁻¹. The present invention, according to oneembodiment, is directed to methods that are useful wherein thepolynucleotide is selected from a group consisting of DNA, RNA, and PNA,and the polynucleotide is single or double stranded.

According to another embodiment of the present inventive methods,intercalating molecules may be removed from polynucleotide molecules, bydelivering a xanthine molecule and forming a xanthine-intercalatingmolecule complex and a polynucleotide molecule. The specific xanthinemolecule and dosage used are dependent upon the circumstances andintercalating molecule or molecules present. Preferably the xanthinemolecule comprises methyl groups at the N1, N3, N7, and/or N9 positions.

Depending on the circumstances and the intercalators present, thexanthine molecule may further comprise preferably oxygen or a halogen(Cl, Br, F, I) at the C8 position. The intercalating molecule isselected from the group consisting of acridine orange, doxorubicin,doxorubicin hydrochloride, novantrone, elipticine, ethidium bromide,Hoeschst 33258, and aflatoxin. The intercalating molecule may be aplanar polyaromatic hydrocarbon molecule. The planar polyaromatichydrocarbon molecule may be benzo(a)pyrene and dimethyl benzathracene.

Additionally, the number of methyl group substitutions on the xanthinemolecule may be increased to five or less.

According to yet another embodiment of the present invention, theinvention provides a composition for the efficient delivery of ananti-neoplastic agent to a patient. Preferably, the composition ismanufactured from an antinoeoplastic agent and at least one carriermolecule. The carrier molecule may be selected from the group consistingof purines, pyrimidines, xanthines, and/or ursic acid. Preferably, thecarrier molecule comprises DNA, RNA, or PNA. The carrier molecule of thepresent invention may be single stranded, double stranded, partiallysingled stranded and partially double stranded, multiple stranded,looped, and/or cross-linked, etc.

The composition of the present invention may, according to oneembodiment, be manufactured from a carrier molecule that containsR-group substitutions around the purine, pyrimidine, xanthine, or uricacid. The composition may also contain R-group substitutions at the N1,N3, N7, or N9 positions. The composition may also contain othersubstitutions at the C2, C4, or C8 positions.

The present invention is useful for the treatment of mammals, humans,birds, fishs, amphibians, yeast, bacteria, nematodes, and insects. Thepresent invention is also applicable to the treatment of plants, fungi,molds, algae, and/or other entities that contain nucleic acids and/orDNA

Compositions of the present invention may be delivered by inhalation,oral administration, topical administration, transdermal administration,IV administration, IP administration, IM administration, byelectroporation, or by liposomes.

In still another implementation of one embodiment of the presentinvention, a method of removing intercalating carcinogenic moleculesfrom a substance that could be ingested by a mammal is provided. Themethod comprises delivering a xanthine molecule to a complex formed byan intercalating molecule and a substance capable of entering a body ofa mammal and complexing with the intercalating molecule. Theintercalating molecule may be an aflatoxin.

In still another implementation of the present invention, methods ofremoving anti-neoplastic intercalating compounds from cellular DNA of amammal are provided. The methods comprise delivering a xanthine moleculeor molecules to the complex formed by the interaction of anintercalating molecule and cellular DNA and forming axanthine-intercalating molecular complex and cellular DNA. Theintercalating molecule may be, e.g., doxorubicin.

Another implementation of the present invention comprises a method forreducing the intercellular concentration of an anti-neoplasticintercalating compounds. The method preferably comprises delivery of axanthine molecule to an intercalating compound. The intercalatingmolecule is preferably doxorubicin.

In still another implementation of an embodiment of the presentinvention, a method of reducing the toxicity to mammals of polyaromatichydrocarbons is provided. The method comprises delivering a xanthinemolecule to the polyaromatic hydrocarbon. The polyaromatic hydrocarbonpreferably is benzo(a)pyrene or dimethyl benzathracene.

Another implementation of the invention is to extact polyheterocyclicdyes and compounds from double stranded DNA. The method comprisescontacting interceptor molecules with the aforementioned dyes andcompounds. Formation of the complex between the polyheterocyclic dyesand interceptor molecules follows the removal of the dye from the doublestranded DNA. The interceptor molecules are preferably purines,pyrimidines, xanthines, porphyrins, uric acid, polynucleotides, orpolymers or mixtures thereof.

In another embodiment, the invention can be used to concentratepolyheterocyclic dyes. The method comprises contacting apolyheterocyclic dye molecule with an interceptor molecule. Interceptormolecules may comprise purines, pyrimidines, xanthines, uric acid,prophyrins, or polynucleotides. In a preferred embodiment, doublestranded DNA is used as the interceptor molecule. Preferably, theconcentration of double stranded DNA should be between 0.001% to 10%(wt/wt).

In still another embodiment of the invention, interceptor molecules areused to concentrate active agents in a medium. The method comprisescontacting interceptor molecules with the agent to be removed. Thechoice of the interceptor molecule depends on the circumstances of theenviroment. Preferably the interceptor molecule may be a pyrimidine,purine, xanthine, uric acid, porphyrin, polynucleotides, or combinationsor mixtures thereof. In another embodiment, the agent to be removed canexist in either a solid, liquid, or gas phase, and may simply becontacted to a support that is in a solid, liquid, or gas phase.

Other features and advantages will become apparent from the descriptionand claims that follow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1-1 shows chemical structures of example representative planarpolynuclear aromatic hydrocarbons.

FIG. 1-2 illustrates chemical structures of selected xanthine compounds.

FIG. 1-3 is a model for a molecular stacking complex of the polyaromatichydrocarbon pyrene and the xanthine 1,3,7,9-tetramethyl uric acid asproposed by DeSantis et al., 1960, 1961.

FIG. 2-1 illustrates tructural diagrams of the DNA intercalator acridineorange and selective xanthines found to form complexes with AO.

FIG. 2-2 is a spectral analysis and K_(assoc) determination for thetheophylline-AO complex.

FIG. 2-3 is an optical absorbance spectra taken from a series oftitrations of chlorophyllin with acridine orange.

FIG. 2-4 shows potential energy curves arising from the resonance of thenative-bond and no-bond electronic states in complexes of xanthines andacridine orange.

FIG. 3-1 is a plot of the optical absorbance spectra of acridine orangeat various concentrations in the presence of and in the absence of 150mM NaCl.

FIG. 3-2 is a plot of a spectrofluorometric analysis of acridine orangeat concentrations ranging from 0.5 μM to 10.0 μM in the absence andpresence of 150 mM NaCl.

FIG. 3-3 shows the optical absorbance spectra of acridine orangetitrated with caffeine.

FIG. 3-4 is an absorbance binding spectra of 3 μM acridine orangetitrated with caffeine.

FIG. 3-5 shows the effect of caffeine and NaCl on the fluorescence ofacridine orange and the formation of the acridine orange-caffeinecomplex.

FIG. 3-6 is an optical absorbance spectra taken from a series oftitrations of 3:M acridine orange with different concentrations ofdsDNA.

FIG. 3-7 shows a delta absorbance plot of 3:M AO titrated with herringsperm dsDNA at DNA concentrations ranged from 30:M to 170:M.

FIG. 3-8 is a plot of a spectrofluorometric analysis of acridine orange[2 μM] with various concentrations of herring sperm dsDNA in thepresence of and in the absence of NaCl [150 mM].

FIG. 3-9 is a plot of a spectrofluorometric analysis of 20 μM dsDNA inthe presence of concentrations of acridine orange ranging from 0.5 μM to10.0 μM, in the presence of and in the absence of 150 mM NaCl.

FIG. 3-10 is a schematic showing the relative population of acridineorange monomers and dimers at concentrations of 2 μM and 20 μM, and thebinding of acridine orange monomers to individual molecules of caffeine.

FIG. 4-1 illustrates example chemical structures of the polyaromaticdsDNA binding agents.

FIG. 4-2 is a photomicrograph of chicken erythrocyte smear stained with10 mM acridine orange at two exposure times to illustrate differentialfluorescence color of nuclear chromatin.

FIG. 4-3 are photomicrographs illustrating the staining of theDrosophila salivary gland polytene chromosome with the DNA minor groovebinder Hoechst 33258 and its subsequent removal by exposure to 100 mMcaffeine for 15 minutes.

FIG. 4-4 shows a change in fluorescence intensity of acridine orangestained chicken erythrocytes rinsed with water, caffeine, andaminophylline over varying periods of time.

FIG. 5-1 is a schematic showing the orientation of caffeine and acridineorange within a polarization bonding complex (from Larsen al., 1996).

FIG. 5-2 plots the absorbance binding spectra of doxorubicin titratedwith various concentration of caffeine.

FIG. 5-3 shows that optical absorbance spatra taken from a series oftitrations doxorubicin with various concentrations of chlorophyllin.

DETAILED DESCRIPTION

The invention is directed, according to one embodiment, to thecomplexation of DNA intercalators with xanthine-based moleculesAccording to one embodiment of the present invention, an excess ofxanthine molecules-is provided such that the intercalator molecules aremore likely to bind to the xanthine molecules instead of othermolecules; e.g., DNA.

According to one embodiment, the DNA intercalator molecule may betreated with xanthine such that the DNA intercalator molecule will bindto the xanthine molecule. The amount of xanthine molecules presentshould preferably be greater than the amount of the intercalatormolecules present, and the xanthine molecule preferably has a sufficientbinding affinity for the DNA intercalator. When DNA is added to thexanthine, and the xanthine has a sufficient binding affinity for the DNAintercalator and an intercalator/xanthine complex is formed, the DNAintercalator will only bind to a minimal extent to the DNA.

The xanthines and purines used in the following examples were obtainedcommercially at a purity of 98% or greater (Aldrich Chemical Company,Milwaukee, Wis.) and were used without further purification. Tetramethyluric acid (1,3,7,9-tetramethyl-8-oxy xanthine) was obtained separatelyfrom ICN Biomedicals Inc., Aurora, Ohio. Acridine orange(3,6-bis[dimethylamino]acridine hydrochloride), >99% purity, (AldrichChemical Company, Milwaukee, Wis.) was prepared in a stock solution at aconcentration of 50 mM in 5 mM HEPES buffer (1.0 M solution, Mediatech,Inc., Herndon, Va.). Acridine orange (AO) was chosen as a representativeDNA intercalator because of its known spectral characteristics and itswide use as a fluorescence chromophore marker for both DNA and RNA(Robinson et al., 1973; Kapuscinski and Darzynkiewicz, 1987, 1990; vonTscharner and Schwarz, 1979). All stock solutions and subsequentdilutions were prepared by dissolving the appropriate weighed amount ofcompound in 5.0 mM HEPES adjusted to pH 7.0 with 0.1 M NaOH. Examples ofthe molecular structure of AO and some selected xanthines found to formcomplexes with AO are presented in FIG. 2-1. The following xanthines areshown in FIG. 2-1: (A) 1-methyl xanthine; (B) 1,3-dimethyl xanthine(theophylline); (C) 1,3,7-trimethyl xanthine (caffeine); (D)1,3,7,9-tetramethyl-8-hydroxy xanthine (tetramethyl uric acid); and (E)3,6-bis [dimethylamino] acridine (acridine orange).

EXAMPLE 1

Example 1 illustrates the complexation of xanthines with an intercalatormolecule. The formation of a xanthine-intercalator complex can bedemonstrated via a color shift using fluorscently labeled intercalatormolecules. A red spectral shift in the optical adsorption maximum of AOupon the addition of a particular xanthine, indicates the possibility ofa complex being formed between the two compounds. Spectral shifts can bemeasured by optical titrations.

Optical titrations (serial additions of various xanthines to a solutionof AO) were performed on a Beckman DU-600 Scanning Spectrophotometer(Beckman Instrument Co., New York, N.Y.) using a 3 mL (1-cm light path)quartz cuvette containing 2.0 mL of a 10 μmol AO solution. The DU600Spectrophotometer was blanked with 2 mL of a pH 7.0 buffered 5 mM HEPESsolution over the full range of wavelengths to be scanned prior tosample analysis. This reduced the need to subtract the backgroundabsorbance from the HEPES buffer. Optical absorbance scans wereperformed over a wavelength range preferably from 400 nm to 550 nm. Theabsorbance spectra at wavelengths longer than 400 nm were minimal forthe xanthines tested. The 10 μM AO solution was scanned before the startof each titration series to quantitatively monitor any changes in theabsorbance spectrum. Optical absorbance experiments were performed usingaliquots varying in volume from 1:L to 20:L taken from prepared xanthinestock solutions (5 mM to 50 mM) and titrated into the cuvette containing2.0 mL of the 10 μM AO solution. The solution in the cuvette was thenthoroughly mixed by 10 seconds of shaking before scanning. Alltitrations were performed at room temperature (25±1° C.). The absorbancespectra were measured in 1 nm intervals and stored in digital form.Digitized scan data was converted to a Lotus Spreadsheet file (*.wk1)using a conversion program supplied by the manufacturer (BeckmanInstruments Inc.) then imported into a Microsoft Excel 97™ Workbook file(*.xls) and saved in this format for further analysis. Spectral data foreach titration was combined and expressed in graphic form (Excel™)(X-axis=wavelength in nm; Y-axis=optical absorbance) for each xanthinetitrated with AO. Initial absorbance data was corrected for the dilutiondue to the addition of the xanthine by multiplying the absorption valueby an adjustment factor determined by the volume of aliquot added(Larsen et. al., 1996). This manipulation slightly increased theabsorption value to offset the effects of dilution. Adjusted absorptionvalues were plotted and compared to spectra from non-complexed AO. Thisallowed for the visualization of an expected spectral shift that occurswhen AO forms a complex with a xanthine.

FIG. 2.2A shows the optical absorbance spectra of the theophylline-AOcomplex in the absence (I) and presence (II) of theophylline. Thespectrum of the theophylline-AO complex is the result of 5 mM oftheophylline and 10 μM of AO in solution and has been adjusted fordilution. The spectral shift (red), shown in FIG. 2-2A, in the opticaladsorption maximum of AO upon the addition of a particular xanthine,indicates the possibility of a complex being formed between the twocompounds. The optical spectrum of AO at 10 μM is displayed in FIG. 2-2Aand exhibits an absorption band in the visible region with a maximum atabout 495 nm. Upon the addition of 1,3-dimethyl xanthine (theophylline)for example, this maximum shifts to about 507 nm. This red shift in theabsorption maximum of AO in the presence of theophylline provides aconvenient method, by using a delta curve (FIG. 2-2B), for determiningthe association constant for the complex formed between AO andtheophylline.

EXAMPLE 2

The kinetics of the system as encompassed in the binding affinity areexplored in Example 2. The association constant, K_(assoc), may be usedto analyze the binding ability of modified xanthines to DNAintercalators. The association constant may be calculated based upon adifferential shift in the absorbance spectrum. When the presence of thexanthine caused a spectral shift, a difference absorbance plot or deltaplot was obtained. Delta plots were constructed by subtracting theadjusted absorption spectrum of AO in the presence of the xanthine fromthe initial spectrum of AO alone at each wavelength and for each aliquotof the titration. This procedure allowed for the creation of differenceabsorption values or delta absorption values. Delta absorption valuesobtained in this manner (Y-axis=delta absorption) were plotted againstwavelength in nm (X-axis). The appearance of a single isobestic pointwas indicative of only two species present in solution, AO, and an AOcomplex. Graphic representation allowed for the selection of awavelength with the maximum sensitivity for determining the associationconstant. This wavelength was characterized by the greatest positiveresponse in delta absorbance values observed upon addition of eachaliquot of xanthine to the AO solution. The AO-xanthine association orbinding constant was determined by the following equilibrium expression:I+X≡IX   EQUATION 1:

Where I represents the intercalator (AO), X represents the interceptormolecule (xanthine), and IX represents the AO-xanthine complex,respectively. This leads to the following equation (see Connors, 1987)for the association constant K_(assoc):ΔA=KD P _(o) [X]/(1+K[X])   EQUATION 2:

Where ΔA is the change in absorption after addition of a xanthine (ΔA %[IX]) and becomes the Y-axis of the delta plot; D is the deltaextinction coefficient (obtained from a fitted curve, as explainedbelow); [X] is the xanthine concentration (moles) in the cuvettefollowing each aliquot addition; P_(o) is the concentration of theintercalator (AO) in moles; and K is the association constant(K_(assoc)). To solve for K, Equation 2 thus becomes:ΔA=K(ΔA _(max))[X]/(1+K[X])   EQUATION 3:

The resulting ΔA values (Y-axis=)A) were plotted against the finalxanthine concentration (X-axis=[X]) at the 8_(max) that exhibited thegreatest sensitivity for monitoring the formation of the AO-xanthinecomplex. The ΔA_(max) value is the maximum absorbance of the complex atsaturation (maximum amount of complex that can be formed at equilibrium)and is equivalent to the delta extinction coefficient multiplied by theconcentration of substrate (i.e. AO) and can be expressed as D[P_(o)].The ΔA_(max) value used to calculate the binding association constant(Equation 3) was iterated from the initial AO spectrum by two approachesas described below.

Equation 3 (ΔA=K(ΔA_(max))[X]/(1+K[X])) is the binding isotherm for a1:1 interaction as represented by the binding of AO with xanthines. Theexperimental data consists of ΔA values obtained as a function of ligand(i.e. xanthine) concentration [X]. The fixed wavelength is chosen togive the largest possible ΔA value. The ΔA_(max) value is the maximumabsorbance that can occur when the substrate (AO) is saturated orcompletely bound to ligand (xanthine). The range of xanthineconcentrations [X] analyzed is listed in Table 1 and should preferablycover at least 60% of the binding isotherm or as much as is permitted bythe experimental situation (Connors, 1987). Percent saturation isdetermined using the formula [K_(assoc)[X]/(1+K_(assoc)[X])]*100. Due tosolubility restrictions by many of the xanthines tested, only fourcompounds exceeded 60% saturation with two compounds at approximately50% saturation. The ΔA_(max) value used to calculate the bindingassociation constant (Equation 3) was iterated from the initial AOspectrum by two approaches.

The first approach to determine K_(assoc) was to select a ΔA_(max) valueestimated from the experimental data followed by experimental iterationof this value to maximize the correlation coefficient of the non-linearanalysis (R²) while not deviating significantly from the model. Thesecond approach for the determination of the K_(assoc) utilized a twoparameter fit where both K and ΔA_(max) were allowed to floatsimultaneously. Results from this second approach yielded mean valueswith standard error ranges for both K_(assoc) and ΔA_(max). Comparisonof the results between the two approaches indicate that either may beused to determine K_(assoc), and in all cases the values obtained forxanthines at approximately 50% saturation and above were notsignificantly different.

Fits to the association curve were determined and a graph created bynonlinear least square analysis with Prism™ software (San Diego,Calif.). The value of the association constant (K_(assoc)) with astandard error was determined from this plot for both approachesdescribed above. Results from the two approaches were compared and theK_(assoc) values having the highest R² values and minimum standarderrors are reported in Table 2-1. In Table 2-1, the compounds aregrouped, on the basis of, increasing numbers of carbonyl groupssubstituted around the purine core structure. The wavelength (λ) in nmof the maximum possible deviation on the delta curve, the square of thecorrelation coefficient (R²) of best fit of delta absorbance data (seetext for equation), and the concentration range over which the xanthinewas analyzed are also shown. A K_(assoc) (M⁻¹) value of zero indicatesno measurable association was detected. The number of resonance statesis listed under “R.S.” with the number of methyl groups indicated inparenthesis at the far right of the table. TABLE 2-1 Results ofspectrophotometric analysis for the determination of binding affinities(K_(assoc)) between acridine orange and substituted xanthines (mean ±S.E.) λ_(max) Concentration K_(assoc) ± S.E. R.S. Compounds (nm) (μM) %Sat.^(a) (M⁻¹) R² (# CH₃) Purine 509  25-2199 *^(b)  ˜2 .9914 2 (0)6-methyl purine —  25-805 —  0 — 2 (1) 6-chloro purine —  10-383 —  0 —2 (0) 1-methyl-6-amino purine 507  25-1247 * ˜14 — 2 (1)3-methyl-6-amino purine 507  25-1507 ˜7   44.5 ± 0.2  .9996 2 (1)O-methyl guanine 511  25-745 *  ˜2 — 4 (1) (2-amino-6-methoxy purine)guanine mono phosphate —  50-455 —  0 — 4 (0) xanthine (2,6-dioxypurine) —  50-475 —  0 — 5 (0) 1-methyl xanthine 511  50-536 * 79.2 ±2.0  .9818 3 (1) 3-methyl xanthine 545  50-762 * ˜10 — 4 (1) 7-methylxanthine —  25-708 —  0 — 4 (1) 1,3-dimethyl xanthine 508  248-1000061.7 157.0 ± <0.1  1.000 2 (2) (theophylline) 3,7-dimethyl xanthine 503 20-412 * 94.5 ± 1.3  .9951 3 (2) (theobromine) 1,7-dimethyl xanthine506  171-10000 61.7 155.3 ± 3.1  .9956 2 (2) 1,3,7-trimethyl xanthine511  495-25000 86.5  255.7 ± 4.8^(c)  .9987 1 (3) (caffeine)1,3-dimethyl xanthine 510  25-1503 47.4 596.2 ± 6.5  .9976 1 (4)ethylenediamine (aminophylline) uric acid (8-oxy xanthine) —  10-338 — 0 — 7 (0) 1-methyl-8-oxy xanthine —  50-1622 —  0 — 5 (1)7-methyl-8-oxy xanthine —  5-305 —  0 — 6 (1) 1,3-dimethyl-8-oxyxanthine 509 1000-5000 16.2 38.5 ± 2.2  .9660 4 (2)1,3-dimethyl-8-chloro xanthine 508  10-5000 67.4 412.5 ± 11.7  .9970 2(2) 1,3,7,9-tetramethyl-8-oxy 511  25-1644 47.6 551.5 ± 9.0  .9945 1 (4)xanthine^(a)The percent saturation of the binding curve as calculated by theformula: K_(assoc) [X]/(1 + K_(assoc) [X]) * 100.^(b)Percent saturation is less than 10%.^(c)As validation of our procedure, the K_(assoc) value of CAF-AOcomplex was found to be 256 M⁻¹ that corresponds closely with the valuereported by both Larsen et al. (1996) and Kapuscinski and Kimmel (1993)of 258 M⁻¹.

In each case where K_(assoc) was determined at xanthine concentrationsbelow approximately 50% saturation of the binding curve, there wasevidence to indicate that if R²>0.99 then the resulting ΔA_(max) valuewas a good prediction of the ΔA_(max) value obtained using data takenover a wider concentration range and at xanthine concentrations >50%saturation. The spectra gave evidence for complex formation between AOand a xanthine even when xanthine concentrations were limited bysolubility and the saturation of the binding curve was substantiallyless than 50%. However, in these cases a binding constant could only bedetermined at a low level of confidence, therefore, such values are notgiven serious weight in the discussion of the results. In oneimplementation, the K_(assoc) value of CAF-AO complex was found to be256 M⁻¹.

As shown in Table 2-1, a methyl substitution at the N1, N3, N7, or N9position can increase the binding affinity for the modified xanthine toDNA. Additionally a halogen at the C8 position also may increase itsbinding affinity to DNA such that the intercalator molecule is lesslikely to bind to DNA. Table 2-1 illustrates that the xanthines shouldpreferably be selected from a group consisting of—methyl xanthine,1,3-dimethyl xanthine, 3,7-dimethyl xanthine, 1,7-dimethyl xanthine,1,3,7-tri-methyl xanthine, 1,3-dimethyl xanthine, 1,3-dimethyl-8-oxyxanthine, 1,3-dimethyl-8-chloro xanthine, and 1,3,7,9-tetramethyl-8-oxyxanthine. In another implementation, the invention may be applied toxanthines with other multiple substitutions on the xanthine molecule

FIG. 2-2B shows the absorbance difference spectra of AO titrated withtheophylline. Spectral analysis was performed on a series of AOtitrations with theophylline. Absorbance values were obtained andadjusted to take into account the effect of dilution. The initialintercalator (AO) concentration is preferably 10 μM in 5 mM HEPES bufferat pH 7.0. Theophylline was pipetted into the AO solution in 1 μlaliquots from a 50 mM stock solution. The resulting plot shows thepresence of a single isobestic point, indicative of only two species insolution. The determination of the association constant, K_(assoc), (theK_(assoc) value between theophylline with AO) is determined from thecurve in FIG. 2-2C. The optical difference spectrum of theAO-theophylline complex in solution displays a single isobestic point,characteristic of only two absorbing species in solution. Using theequation ΔA=K(ΔA_(max))[X]/(1+K[X]) (see Equation 3), the calculatedK_(assoc) value for the AO-theophylline complex is about 157.0±<0.1 M⁻¹.The other xanthines were similarly analyzed and their K_(assoc)constants are reported in Table 2-1.

Of the twenty-two different xanthines tested, six were determined tohave substantial (∃150 M⁻¹) association constants. An additional eightcompounds showed some evidence of complex formation with AO. These 14compounds are listed in Table 2-1 along with the wavelength (nm) atwhich the binding constant was derived, the concentration range of thexanthine tested (μM), the percent saturation of the binding curve, theK_(assoc) (M⁻¹) value, the regression analysis R² value, the number ofresonance states “R.S.” (discussed later), and the number of substitutedmethyl groups (# CH₃). None of the seven purines tested revealed anysubstantial binding affinity with AO.

In another implementation, compounds that were found to have substantialbinding with AO had three common structural similarities: (1) the N₁ orN₃ on the xanthine core structure must be substituted with a methylgroup, (2) oxygen or chlorine substitution at C₈ increased K_(assoc)when the number of resonance states remained unchanged, and (3)K_(assoc) increased with increase in number of methyl groupsubstitutions, generally with an empirical fit (R²=0.99) to a secondorder polynomial equation.

Compounds without a methyl group substitution such as xanthine and8-hydroxy xanthine (uric acid) did not complex with AO, as determined byoptical titration. Of the six compounds that exhibited significantK_(assoc) values, all had a methyl group substituted at N₁ and carbonylgroups at C₂ and C₆. Alternatively, compounds without a methyl group atN₁ of the xanthine base structure, but having substituted methyl groupsin other positions (3-methyl xanthine, 7-methyl xanthine, 3,7-dimethylxanthine, and 7-methyl-8-oxy xanthine) showed little or no affinity tocomplex with AO except for 3,7-dimethyl xanthine (theobromine). Thecompounds 1-methyl xanthine, 1-methyl-8-oxy xanthine, 1,3-dimethyl-8-oxyxanthine, and 1,3-dimethyl-8-chloro xanthine have carbonyl groups at C₂and C₆ and a methyl group substituted at N₁, yet failed to exhibit anysignificant binding affinity for AO. One difference between 1-methylxanthine, which has a binding affinity for AO of 79.2±2.0 M⁻¹, and1-methyl-8-oxy xanthine, is a carbonyl group at C₈. Likewise, onedifference between 1,3-dimethyl xanthine (theophylline) with a bindingconstant of 157.0±<0.1 M⁻¹ and 1,3-dimethyl-8-chloro xanthine or1,3-dimethyl-8-oxy xanthine is the presence of an atom with highelectron affinity (oxygen or chlorine, respectively) substituted at C₈.The presence of a carbonyl group at C₈ significantly reduces the bindingability of mono- and di- methyl substituted xanthines except for thetetramethyl substituted TMU, which exhibits a lower binding affinitythan aminophylline; and also contains a total of four methyl groups.

It is possible that the binding of AO to substituted xanthines can bemodified by specific substitutions in systematic ways as discussedbelow. Additionally, the formation of a complex between AO and xanthinederivatives may be predicted based on the theoretical energy state ofthat particular xanthine. Finally, it is likely that a correlationbetween K_(assoc) and the ability of some xanthines to increasesolubility of another PAH, benzo(a)pyrene, and other potentialapplications. It is noted that the invention is not limited in any wayby the proposed mechanisms.

The formation of a complex between any two molecules occurs preferablywhen it is thermodynamically advantageous for the complex to exist(Wallwork, 1961). Since the formation of a complex allows for areduction in the overall energy state of a molecule, it would be lessthermodynamically advantageous for a lower energy molecule to form acomplex than for a higher energy molecule. Thus, as the energy of amolecule decreases, the probability of that molecule forming a complexalso decreases. The overall energy state of the xanthine molecule, and,therefore, its potential to form a complex with AO, probably is due tothree important factors: (1) the number of potential resonance states,(2) the amount and position of N-methylation, and (3) the chemicalcharacteristics of the R-group substituted at the C₈ position of thexanthine ring. All of these factors can influence π-orbital chargedistribution, the hydrophobicity of the molecule, the electronic energystate, and, therefore, the thermodynamic potential for the formation ofa complex.

Resonance states occur within molecules when, quantum-mechanically, theyare able to exist simultaneously in different structural forms thatde-localize the electronic density. This can occur with a “floating”double bond as seen in the five membered ring in xanthine, or with adouble bond that is in equilibrium with a single bond as in tautomericcompounds such as those formed by uric acid. When the resonance statesfor the molecules tested were counted, those with only one resonancestate were found to have the higher K_(assoc) values (see Table 2-1).Conversely, those molecules with four or more resonance states exhibitedlittle or no complex formation. Examination of the K_(assoc) values forboth substituted xanthines and uric acids versus the number of resonancestates (Table 2-1) shows that as the number of resonance statesincreases, K_(assoc) values decrease. Among the di-methyl substitutedxanthines, 3,7-dimethyl xanthine with three potential resonance stateshas a 41% lower K_(assoc) value than 1,7- or 1,3-dimethyl xanthine, bothof which have only two potential resonance states. The lower K_(assoc)value for 3,7-dimethyl xanthine suggests that there is a lower energystate associated with the higher number of potential resonance states.Both 1,3-dimethyl xanthine and 1,7-dimethyl xanthine have two potentialresonance states and have a similar K_(assoc) value. Structurally bothmolecules differ only by the position of the second substituted methylgroup on the xanthine core, either at the N₃ or N₇ position, suggestingthat the position of the second methyl substitution contributes littleif any to the stability of the complex. Additionally, the difference inK_(assoc) between 1,3-dimethyl xanthine and 1,3-dimethyl-8-chloroxanthine, both with only two potential resonance states, must be due tothe difference in the chemical characteristics between hydrogen andchlorine. The number of potential resonance states in which a moleculecan exist in addition to the chemical characteristics of the R-groupsubstituents, probably exert a substantial influence on the energy stateof the xanthine molecule. Consequently, the position of the substitutionon the ring structure should have only a minor contribution if thenumber of resonance states is not changed by a shift in the position ofthe R-group.

One way to increase the electron-donor properties of purines orxanthines is N-methylation. The degree to which such methylationincreases these properties depends in part on the chemical structureand, for a given compound, on the position of the alkyl group. Based onmolecular orbital theory, the position of N-methylation of the xanthinecore structure should increase the electron-donor properties withN₃>N₇≅N₉>N₁ In uric acid however, the activation of its electron-donorproperties should occur for N-methylation at N₇>N₃>N₉>>N₁. As seen inTables 2-1 and 2-2, a pattern increasing K_(assoc) values betweensubstituted xanthines and AO occurred with increasing N-methylation.With each addition of a methyl group to the xanthine core structure,K_(assoc) approximately doubles. Binding affinity (K_(assoc)) versusmethyl group substitution was plotted using the exponential formula Y=Start*exp(K*X) where the plot begins at Y=Start and increasesexponentially with rate constant K with a doubling equal to 0.69/K.Regression analysis resulted in no significant deviation from theexponential equation (P=0.429) with an R² value of 0.99. Thus, a highcorrelation exists between the number of methyl substitutions on thexanthine ring and the resulting K_(assoc) value.

The results illustrated in Table 2-1 indicate that binding affinity(K_(assoc)) is also affected by the position of the methylated nitrogensaround the xanthine core. Among the mono-methylated xanthines, both1-methyl xanthine and 3-methyl xanthine were found to exhibit someevidence of weak binding affinity to AO as determined byspectrophotometric analysis, but the binding was not definitive (i.e.,K_(assoc) <150 M⁻¹ and limited concentration range i.e., saturation<10%). However, a single methyl substitution at N₇ resulted in nodetectable evidence for AO complexation (saturation <10%). Examinationof the xanthine core structure shows that the N₁ position on the ring issituated between two double bonded oxygen groups, whereas N₃ is nearonly one double bonded oxygen at C₂, while N₇ has no double bondedoxygen nearby. The electron directing effect of a methyl group maylikely enhance the resonance between the lone-pair N₇ p-orbitalelectrons and the neighboring oxygen groups. This in turn couldcontribute to the electron density of the π-electron cloud in thevicinity of the ring structure. Thus, the contribution to the formationof π-π bonds between AO and the mono-methyl-substituted xanthines may beenhanced by this nearest neighbor effect, with methyl substitutionhaving the greater effect at N₁>N₃>N₇≅N₉. There is probably somecontribution to the stability of the complex due to the elevated energystate of the core molecule through the loss of two resonance states, inaddition to the electron donor contribution from the methyl group.

In the case of 1,3-dimethyl xanthine ethylenediamine (aminophylline),there are two theophylline groups linked by ethylenediamine.Aminophylline exists in solution as two molecules of 1,3-dimethylxanthine (theophylline), each molecule bonded ionically at the N₇position of the xanthine core to the nitrogen at each end of theethylenediamine linker molecule. The chemical structure of aminophyllinein solution can thus best be described as 1,3-dimethyl xanthine with anethylenediamine theophylline R-group ionically bonded at the N₇position. The resulting K_(assoc) value suggests that the loss of aresonance state, together with the increased electron-donor capacity ofthe R-group at N₇, significantly enhances the ability of theophylline tocomplex AO by 280% as compared to 63% enhancement with the substitutionof a methyl group at N₇ in the case of caffeine (1,3,7-trimethylxanthine).

The influence of the number of methyl groups on K_(assoc) is lessobvious when there is a simultaneous substitution of either oxygen orchlorine at the C₈ carbon. As can be seen in Table 2-1, the substitutionof a carbonyl group at C₈ significantly decreases the ability of AO tobind with methyl-substituted xanthines (e.g., K_(assoc) for1,3-dimethyl-8-oxy xanthine is 38.5 M⁻¹). By examination of FIG. 2-1, itcan be seen that in the xanthine core structure, the double bond at C₈can resonate between N₇ and N₉ and the hydrogen substituted carbon atC₈. With a carbonyl substitution at C₈, resonance of the double bondbetween C₈ and N₇ or N₉ is enhanced and allows for an additionalresonate state to exist when the oxygen at C₈ tautamerizes between thecarbonyl and the hydroxyl form. Simultaneously, the dipole moment of themolecule has significantly decreased and shifted away from thesix-membered ring toward the oxygen at C₈, becoming more centralizedover the core structure. With the substitution of chlorine at C₈, thedouble bond can still resonate between N₇ and N₉, but the π-electroncloud will be less centered above and below the planar rings of thexanthine and, therefore, will be displaced toward the chlorine atom withits higher electron affinity. These features appear to enhance K_(assoc)about 10-fold. It should be noted that the addition of methyl groupsubstitution at N₇ and N₉ tends to offset the electron affinity effectsof the carbonyl at C₈, and the resulting change in the dipole moment.Therefore, the substitution of a carbonyl group in the C₈ positioncreates a mobile or resonate hydrogen (between N₇ and N₉) that isprobably not present in the tri-methyl xanthine or the tetramethyl uricacid molecules.

In the case of the AO-caffeine system, the spectral change is quitesimilar to that observed for the AO-DNA intercalation complex,suggesting the occurrence of comparable interaction between thechromophore and the hydrophobic surface of the aromatic compounds suchas caffeine or DNA base pairs. The monomerizing (disaggregating)capacity of weakly polar solvents, α-cyclodextrin, and detergentsoriginates from the reduction of the degree of dye solvation. Underthese conditions, hydrophobic interactions between stacked dyechomophores (dimers) can generally disappear and absorption spectracharacteristic of the dye monomers are obtained.

Organic solvents, as well as the internal space of α-cyclodextrin anddetergent micelles, provide the same hydrophobic weakly polarenvironment that occurs in the intercalative cavity of nucleic acids.The simplest formulation, hydrophobic interactions between DNA, AO, andplanar xanthines can be represented as stacked complexes.

Table 2-2 compares the K_(assoc) values found in Table 2-1 with thesolubility values reported by Weil-Malherbe (Weil-Malherbe, 1946a,1946b). TABLE 2-2 Relationship between: number of methylations on thexanthine ring, the K_(assoc) value with acridine orange (this report)and the ability of the xanthine compound to solubilize benzo(a)pyrene inwater as reported by Weil-Malherbe (1946a, 1946b). Compounds CH₃K_(assoc) (M⁻¹) Solubility 1-methyl xanthine 1  79^(a)  17^(b)1,7-dimethyl xanthine 2 155  27 1,3-dimethyl xanthine 2 157  281,3,7-trimethyl xanthine 3 256 100 1,3,7,9-tetramethyl-8-oxy xanthine 4552 524^(a)Percent saturation of the binding curve was less than 10 percent.^(b)Due to lack of a specific solubility value for 1-methyl xanthine,the average of the solubility values reported for 3-, 7-, and 9-methylxanthine was used.

In Table 2-2, solubility units are based on a comparison to caffeine,which was given a value of 100. A non-linear regression analysis revealsa good fit to the exponential equation Y=Start*exp(K*A) between thenumber of methyl groups substituted on the xanthine ring and thesolubility values reported by Weil-Malherbe (1946a, 1946b) for xanthineswith benzo(a)pyrene (R²=0.98). A comparison of the results from both theK_(assoc) values and the solubility values from Weil-Malherbe shows asignificant linear correlation (P=0.002, R²=0.90).

The effect of structural changes on the interaction of xanthines with AOfollow closely those described by Weil-Malherbe in his studies of thesolubilization of polyaromatic hydrocarbons with purine bases. One ofthe structural differences among a purine, hypoxanthine, xanthine, anduric acid core molecule is the number and presence of oxygen groupsbonded to a carbon atom at C₂, C₆, or C₈ of the purine base structure.Purine has no substituted oxygen groups whereas hypoxanthine has anoxygen group substituted at C₆, xanthine has an oxygen at C₂, and C₆,and uric acid has an oxygen at C₂, C₆, and C₈. Weil-Malherbe observedthat the solubilization of the PAH benzo(a)pyrene increased withincreasing N-methylation and, up to a point, with increasingsubstitutions and increasing electron affinity of the R-groups at C₂,C₆, or C₈ around the purine ring. He found that increasing the number ofcarbonyl groups around the purine ring increased the solubility ofbenzo(a)pyrene in all cases. However, the substitution of a chloro-,thio-, or amino-group might either increase or decrease solubility. Therelationship between N-methylation (X-axis) and solubilization (Y-axis)can be fitted, empirically, with the exponential equationY=Start*exp(K*X) (R²=0.98).

This relationship suggests that the solubilization of PAHs such asbenzo(a)pyrene, may be due to complex formation between the PAH and thexanthine, and, therefore, directly related to K_(assoc). Table 2-2compares the K_(assoc) values and the solubility values fromWeil-Malherbe (1946a and 1946b) and reveals the existence of asignificant linear correlation (P=0.002, R²=0.90) between the two setsof data values. Based on these results, solubilization can be used toscreen potential interceptor molecules for their ability to form π-πstacking complexes with DNA Rapid screening of potential interceptormolecules for their ability to form π-π bonding complexes with DNAintercalators, could be achieved using Bowman and Beroza (1965a, 1965b,1965c, 1966a, 1966b) partitioning values (P-values) utilizing the methoddescribed by Robbins (1979).

Knowledge of the interaction of selected xanthines with PAHs haspractical applications. The interaction of xanthines such as caffeinewith the anti-neoplastic drug doxorubicin (DOX) causes the reduction ofblood plasma levels of DOX and a simultaneous increase at theintracellular level. The ability to optimize the binding affinity orK_(assoc) value between two interacting classes of compounds wouldcreate novel approaches relevant to drug therapy and delivery. Forexample, enhancing target site concentration of an anti-neoplastic drugsuch as doxorubicin could simultaneously increase cellular toxicitywithin the tumor while decreasing the overall systemic toxicity by areduction in blood plasma levels. Additionally, interceptor moleculescould potentially be designed to bind with specific carcinogens with avery high affinity and therefore, significantly reduce their biologicaleffects. Finally, by using the laws of mass action, on whichpolarization bonding depends, the possibility exists for the creation ofcompounds for the selective prevention and/or reversal of DNAintercalation by PAHs.

Results from spectral analyses of various xanthines with theDNA-intercalator AO, demonstrate that specific xanthines can complexwith AO. This interaction is attributed to a type of B-B electronorbital interaction known as polarization bonding. Xanthines withspecific R-group substitutions modify binding affinity to AO in at leastthree systematic ways: (1) increased with an increase in N-methylationaround the xanthine core, (2) increased by an increase in the potentialenergy of the xanthine molecule, as evidence by a decrease in the numberof resonance states, and (3) the electron affinity and other chemicalcharacteristics of substituents appears to affect K_(assoc). Thedominant influence in the formation of such complexes appears to be vander Waals interactions resulting in maximal B-orbital overlap betweenthe two molecules of the complex, and the hydrophobic effects of themethyl group substitutions. Additionally resonance with a donor-acceptorcharge transfer state may also contribute to the stabilization of thecomplex. The hydrophobic effect is an entropic rather than an enthalpicphenomena based on the exclusion of water molecules. These resultssuggest a possible role for CAF and other xanthines as interceptormolecules in cancer therapy.

These application of this invention is not limited to the DNAintercalator acridine orange. The invention may be applied to otherheterocylic compounds such as doxorubicin and its associated salts,novatrone, elipticine, ethidium bromide, Hoeschst 33258, aflatoxin, orother similar molecules. In other implementation, polanar polyaromaticcompounds such as benzo(a)pyrene and dimethly benzathracene may be used.Moreover, the invention is not limited to double stranded DNA. Othercompounds such as RNA, PNA, and single and double stranded versionsthereof, may be used.

The invention is not limited to the use of xanthines as interceptormolecules. For example, chlorophyllin and porphyrins may be used as abinding agent to intercalator molecules. FIG. 2-3 shoes the opticalabsorbance spectra titrating cholorphyllin with increasingconcentraitons of acridine orange. The selected spectra denotingimportant transitions within the titration series are denoted by thebold lines. At AO:chlorophyllin ratio between 0.1 and 30, prevalentpeaks occur at λ_(abs) of 415 nm for chlorophyllin, 475 nm, and 492 nm.These later peaks correspond to the prevalent absorbance peaks found atconcentrations of AO at 10 μM and higher concentrations. This figureillustrates that chlorophyllin complexes with acridine orange. Thus,other compounds may be used as interceptors such as, but not limited toporphyrins, purines, pyrimidines, polynucleotides and uric acid.

EXAMPLE 3

The addition of salt affects the conformation of both the polynucleotideand the intercalator molecule. Thus, the use of modified xanthines maybe tailored through the control of the ionic environment. As in theprevious examples, the ability of the modified xanthines to bind with anintercalating molecule is described via binding affinities.

Association constants were calculated using a modification of anequation described by Zaini et al. (1999) to determine equilibriumconstants for weakly bound complexes by absorbance spectrophotometry. Bydefinition K=X_(d)/(X_(m* X) _(m)), where K is the binding constantK_(assoc), X_(d) is the concentration of the AO dimer and X_(m) is theconcentration of the AO monomer. Conservation of mass givesX_(t)=X_(m)+2*X_(d), where X_(t) is the total concentration of AO insolution. Rearranging the conservation of mass equation givesX_(m)=X_(t)−2*X_(d), and substituting into the equation for K_(assoc)and rearranging gives Equation 4:K _(assoc)(X _(t) *X _(t)−4*X _(t) *X _(d)+4*X _(d) *X _(d))−X_(d)=0  EQUATION 4:

Rearranging gives:X _(d) *X _(d)−(X_(t)+1/(4*.K _(assoc))*X_(d) +X _(t) *X _(t)/4=0  EQUATION 5:

Applying the quadratic formula (X=[−B±SQRT(B²−4AC]/2A) to Equation 3-5and taking a physically reasonable root gives Equation 3-6:X _(d) =[X _(t)+1/(4*K)−SQRT(X_(t)/(2*_(K))+1/(16*K*A))]/2   EQUATION 6:

Where SQRT ( ) indicates taking the square root of the content of theparentheses. The fluorescence values at λ_(em) 530 nm (F₅₃₀) is afunction of X_(t) and F₅₃₀=X_(d)*ε, where “ε” is the fluorescence yieldof the dimer. The equation was solved by non-linear least squares usingPrism™ software and binding constants determined. Concentrations of thereactants were adjusted for dilution whereas AO concentrations arereported without adjustment for dilution. Fluorescence values (f_(i))are reported as raw data normalized to scale and not adjusted by adilution factor following aliquot additions. Absorbance data taken at468 nm was also used to calculate the K_(assoc) for the AO-AO dimerusing Equation 6.

In still another implementation, AO is known to exhibit two λ_(max)fluorescence peaks, depending upon the type of interaction it has withitself or other compounds. For example, when AO intercalates into DNA,presumably by forming a B-B interaction or stacking complex, it has acharacteristic green fluorescence maximum at about λ_(em) 526 nm.Alternatively, when AO associates with other compounds electrostatically(ionic) through charge transfer it has a characteristic fluorescencemaximum at about λ_(em) 650 nm. The two emission wavelengths, λ_(em) 635nm and λ_(em) 530 nm, represent the approximate λ_(max) for thefluorescence emission of AO when electrostatically associated with othercompounds (635 nm) and in a stacked (B-B) association with itself oranother compound or compounds (530 nm).

Optical absorbance of AO at various concentrations indicates that AOdissolved in 5 mM HEPES solution buffered at pH 7.0 has two spectralpeaks (FIG. 3-1A). In the example shown in FIG. 3-1A, absorbance wasscanned between 400 nm and 550 nm over a range of AO concentrations from2.5 μM to 20 μM in a 5 mM solution of HEPES buffer at pH 7.0. Theconcentration of AO for each spectrum is indicated in the legend bynumber. Note that the absorbance at 492 nm (A₄₉₂) is indicative of theconcentration of the AO monomer, whereas the absorbance at 468 nm (A₄₆₈)is indicative of the concentration of the AO-AO dimer (Kapuscinski andKimmel, 1993). AO at a concentration of 2.5 μM exhibits a distinctspectrum with a λ_(max) of 492 nm. At increasing concentrations of AO, asecondary spectral peak at 468 nm became more distinct and increased inamplitude. When the ratio of the change in absorbance value at 492 nmover the change in absorbance values at 468 nm was plotted versus AOconcentrations ranging from 2.5 μM to 20 μM a significant (R²=0.99)linear correlation indicating a relationship between the formation of AOdimers as a function of AO concentration resulted.

The example in FIG. 3-1B shows the same optical absorbance of AO atvarious concentrations under the same conditions except with addition of150 mM NaCl. The absorbance was scanned between 410 nm and 540 nm over arange of AO concentrations from 10 μM-45 μM in a 5 mM solution of BEPESbuffer at pH 7.0 in the presence of 150 mM of NaCl. The concentration ofAO for each spectrum is indicated by arrow. Note that the magnitude ofthe absorbance at 492 nm (A₄₉₂) decreases in relation to the absorbanceat 468 nm (A₄₆₈), as a function of AO concentration. Thus, the presenceof 150 mM NaCl in solution causes an overall decrease in the rawabsorbance values. In addition, the presence of a physiologicalconcentration NaCl significantly increased the absorbance peak at 468 nmindicating an increase in the concentration of AO dimer in the presenceof NaCl.

The fluorescence characterization of AO at low concentrations [0.5-10 μMis illustrated in example of FIG. 3-2A, wherein the spectrofluorometricanalysis was performed at λ_(ex) 485 nm and emission was observed atλ_(em) 530 nm and λ_(em) 635 nm. The error bars shown standard deviationunless obscured by legend symbols (n=4). The results also indicate theeffect of a physiological concentration of NaCl [150 mM] on thefluorescence of AO at both λ_(em) 530 nm (green) and λ_(em) 635 nm (red)over a range of AO concentrations from 0.5 μM to 10.0 μM. At an AOconcentration of between 5-6 μM, the amplitude of green fluorescence atλ_(em) 530 nm increases abruptly. The red fluorescence (λ_(em) 635 nm)also increases abruptly between 5-6 μM. However, the magnitude of theincrease at λ_(em) 635 nm is proportionally less than that found atλ_(em) 530 nm. As the concentration of AO increases from 5 μM to 6 μM,the mean fluorescence values were changed from 457±16 to 5,197 ±289 atλ_(em) 530 nm and from 174±4 to 1,371±41 at λ_(em) 635 nm (n=4). At AOconcentrations from 0.5 μM to 5 μM, the addition of NaCl [150 μM]significantly increases green fluorescence at λ_(em) 530 nm, whereas nosignificant change in the mean fluorescence values was observed atλ_(em) 635 (see FIG. 3-2B). The example shown in FIG. 3-2B depicts thebinding plot calculated at λ_(em) 530 nm for the molecular associationof AO with itself in the absence of and in the presence of NaCl. Theassociation constants (K_(assoc)) for AO derived from these two plotsare 118,000±16,120 M⁻¹ (R²=0.98) in the presence of NaCl [150 mM] and37,410±4,758 M⁻¹(R²=0.97) in the absence of NaCl. Error bars denotestandard deviation unless hidden by legend symbols (n=4). The presenceof 150 mM NaCl has no significant effect on the fluorescence intensityof AO at either λ_(em) 530 nm or λ_(em) 635 nm at AO concentrations of 6μM and above.

Association constants were calculated for AO self-association byassuming the fluorescence values derived at λ_(em) 530 nm reflect theextent of dimer formation. Association constants (K_(assoc)) werecalculated from the change in mean fluorescence values (deltafluorescence) at λ_(em) 530 nm (see FIG. 3-2B). At AO concentrationsfrom 0.5 μM to 5 μM, K_(assoc) was determined to be 37,410±4,758 M⁻¹(R²=0.97) in the absence of NaCl and 118,000±16,120 M⁻¹ (R²=0.98) in thepresence of 150 mM NaCl (see FIG. 3-2B). For AO concentrations >5 μM,the association constants were determined to be 46,700±1,807 M⁻¹(R²=0.99) for AO alone and 41,820±3,671 M⁻¹ (R²=0.99) for AO in thepresence of NaCl. This indicates a significant AO concentrationdependent difference in the association constant in the presence of 150mM NaCl at low AO concentrations (<5 μM) but not a significantdifference at AO concentrations above 5 μM.

Results of the optical titration of 10 μM AO with CAF over a range of 0to 4.5 mM are depicted in FIG. 3-3. The spectrophotometric analysisshown in the example of FIG. 3-3 was performed on a 10 μM solution of AOin 5 mM HEPES buffered at pH 7.0. CAF was titrated into the cuvettecontaining the AO solution in aliquots of 20 μl taken from a 50 mM stocksolution, thus increasing the CAF concentration in increments of 0.5 mM.Absorbance values were corrected to account for dilution. The initialspectrum of 10 μM AO in the absence of CAF (A) gave a λ_(max) of 492 nm.With increasing concentration of CAF, the λ_(max) of the AO-CAF spectrumshifted to 500 nm at a final CAF concentration of 4.5 mM (B).

The spectrum shown in FIG. 3-3 exhibits a maximum absorption band in thevisible region at 492 nm. Upon addition of CAF, the optical absorbancemaximum of AO shifts from 492 nm to 500 nm. This red-shift provides thebasis for a method to determine the association constant (K_(assoc)) forthe complex formed between AO and CAF. This method is demonstrated inFIG. 3-4, which displays the optical absorbance difference spectrum(delta absorbance) of the AO-CAF complex. The delta absorbance valuesfor the example shown in FIG. 3-4 were taken at λ_(max) 511 nm. Note thepresence of a single isobestic point at 500 nm. The inset shows a plotof CAF concentration versus delta absorbance depicts the binding curvefor the formation of the AO-CAF complex. To determine the associationconstant for the AO-CAF complex, delta absorbance values were taken atλ_(max) 511 nm and plotted against the concentration of CAF. The deltaabsorbance spectrum displays a single isobestic point characteristic ofonly two absorbing species in solution and a maximum deviation peak atλ_(max) 511 nm. The calculated K_(assoc) for the AO-CAF complex wasfound to be 255±5 M⁻¹ (R²=0.997) and a 1:1 stoichiometry. The AO-CAFbinding plot depicted in the INSET to FIG. 3-4 fits well to a model inwhich AO and CAF form a 1:1 complex.

Spectrofluorometric analysis was performed at an AO concentration of 2μM to determine the effect of CAF and a physiological concentration ofNaCl on the fluorescence spectra of AO. CAF was added over a series ofCAF:AO molecular ratios ranging from 2500:1 to 0.5:1 and thefluorescence intensity determined at λ_(em) 530 nm and 635 nm. Thefluorescence intensity was again determined following the addition ofNaCl to a final concentration 150 mM and pH 7.0 to each test wellcontaining both AO and CAF.

The data in FIG. 3-5A depicts the mean fluorescence values at λ_(em) 530nm of 2 μM AO with CAF in the absence of, and in the presence of, 150 mMNaCl over a range of CAF to AO molecular ratios from 0.5 to 10.0 (1 μMto 20 μM CAF). The example shown in FIG. 3-5A is a plot of fluorescenceintensity of AO excited at λ_(ex) 485 nm and monitored at λ_(em) 530 nmversus CAF concentration to show the effect of CAF and NaCl on thefluorescence of AO and the formation of the AO-CAF complex. Solutions of2 μM AO were analyzed using spectrofluorometric techniques in thepresence of various concentrations of CAF with and without addition ofNaCl [150 mM. Error bars indicate standard deviation unless hiddenbehind the symbol (n=3). The initial fluorescence values for AO at aconcentration of 2 μM with and without 150 mM NaCl differedsignificantly prior to titration with CAF. Specifically the presence ofNaCl in the 2 μM AO solution significantly increased the fluorescencevalue at λ_(em) 530 nm from a mean of approximately 400 to approximately500. However, upon the addition of increasing amounts of CAF the meanfluorescence values increased significantly over the initial meanfluorescence values. The addition of 150 mM NaCl to the series of AO-CAFsolutions did not significantly effect (p>0.05) the mean fluorescencevalues over the full range of CAF concentrations tested up to a CAF:AOratio of 1000. However at the CAF concentration of 5 mM (molecular ratio2,500:1) there was a significant change in the mean fluorescenceintensity with the value going from 1,245±53 for CAF and AO alone to1,150±34 with the addition of 150 mM NaCl. The gradual change influorescence intensity at λ_(em) 530 run went from a mean value of 671±109 (n=3) at a CAF:AO ratio of 0.5 (1 μM CAF) to a value of 1,245±53 ata molecular ratio of 2,500 (5 mM CAF). The mean values in fluorescenceintensity that resulted with the addition of either CAF or CAF with NaClto AO [2 μM] did not significantly differ from each other in meanfluorescence values at λ_(em) 635 nm for most test conditions.

Mean spectrofluorometric values were plotted against CAF concentration(see FIG. 3-5B) to determine an association constant (K_(assoc)) for theAO-CAF complex in the absence of and in the presence of 150 mM NaCl. Theexample association curves of FIG. 3-5B were plotted from netfluorescence values (minus background fluorescence) versus CAFconcentration. This delta fluorescence plot was used to determineK_(assoc) for the AO-CAF complex at an AO concentration of 2 μM in theabsence and presence of 150 mM NaCl. K_(assoc) for the AO-CAF complex at2 μM AO was found to be 254±27 M⁻¹ (R²=0.89) in the absence of 150 mMNaCl and 216±20 M⁻¹ (R²=0.93) in the presence of NaCl. The associationconstant for the formation of the AO-CAF complex at an AO concentrationof 2 μM was determined to be 254±27 M⁻¹ and 216±20 M⁻¹ in the absence ofand in the presence of NaCl respectively. These mean value differencesare statistically significant.

Experiments were undertaken to characterize the interaction of AO withDNA over a range of DNA base pairs (bp) to AO ratios in the absence of,and in the presence of, a physiological concentration of NaCl. As theratio of bp to AO increased from approximately 0.4 to 0.65, an overalldrop in absorbance values between the wavelengths of 450 nm and 525 nmoccurred (FIG. 3-6). The example shown in FIG. 3-6 is a dsDNA bp: AOratio of selected spectra denoting important transitions within thetitration series are denoted with bolder lines. Some of the spectralseries is not shown to simplify the data presentation. At bp: AO ratiosbetween 0.0 and 0.75, prevalent peaks occur at 8_(abs) of 468 nm and 492nm. These peaks correspond to the prevalent absorbance peaks found atconcentrations of AO at 10:M and higher concentrations. The absorbancespectra from bp: AO ratios above 0.75 denote a definitive red-shift inabsorbance with the simultaneous creation of two distinct peaks, one at475 nm and the other at 503 nm.

With continued additions of aliquots of DNA, the absorbance peak near468 nm is still apparent while the initial main absorbance peak at 492nm is minimally detectable. Additionally, with the continued additionsof aliquots of DNA and the subsequent increase in the bp:AO ratio above0.65, the absorbance at λ_(abs) 475 nm began to increase and to displaya red shift from 468 nm to 475 nm. This spectral peak at λ_(abs) 475 nmgradually increased in height with increasing DNA concentration beforereaching an absorbance plateau at a bp:AO ratios of 3.0 and above.Additionally, at bp:AO ratios of about 1.72. The absorbance peak nearλ_(abs) 503 nm increased in amplitude with each additional aliquot ofDNA. This absorbance at λ_(abs) 503 nm steadily increased throughout therange of titrations from a bp:AO ratio of 1.72 to 9.13. Table 3-1 is asummary and description of the optical absorbance peaks observed duringthe titration of 100 μM AO with dsDNA and the range of bp:AO molecularratios over which these absorbance peaks were observed during thetitration series. The data reported in Table 3-1 were confirmed withadditional serial titrations of AO at 3 μM, 5 μM, 10 μM, and 20 μM. Inanother example, titrations at AO concentrations of 3 μM, 5 μM, 10 μM,and 20 μM yielded similar results. TABLE 3-1 Summary and description ofoptical absorbance maxima peaks observed during the titration of 100 μMacridine orange (AO) with dsDNA and the range of bp:AO molecular ratiosover which these absorbance peaks were observed during the titrationseries. λ_(max) Absorbance bp:AO Ratio 468 nm <0.09-2.99 475 nm 1.72-9.13 492 nm <0.09-0.53 503 nm  0.97-9.13

Spectral data from the titration of 3 μM AO with dsDNA was used togenerate the delta absorbance plot shown in FIG. 3-7. The resultsindicate a single isobestic point at 470 nm. The association constantwas calculated from a plot of delta absorbance values at 504 nm versusDNA concentration (see inset FIG. 3-7). The DNA concentrations rangedfrom 30:M to 170:M. The K_(assoc) from this plot was 37,660±431 M⁻¹.

In another implementation, a spectrofluorometric analysis of theinteraction of AO with dsDNA at λ_(ex) 485 nm and λ_(em) 530 nm and 635nm was also conducted. The interaction of DNA with lower concentrationsof AO [2 μM] mimicked the results found at the higher AO concentrationof 20 μM for bp:AO ratios of 4:1 and above. The association curve for AOover a series of steadily increasing DNA concentrations ranging from 8to 250 μM of DNA is shown in FIG. 3-8.

The example shown in FIG. 3-8 illustrating the effect of NaCl on thefluorescence of 2 μM AO was determined over a range of bp:AO ratios from4 to 62.5. The excitation wavelength was 485 nm while emission wasmonitored at 530 nm. The association constant for 2 μM AO with DNA wasdetermined to be 31,990±310 M⁻¹ (R²=0.98) in the absence of NaCl and31,690±498 M⁻¹ (R²=0.96) in the presence of NaCl. Error bars are shownto denote confidence intervals at 95% unless hidden by a data pointsymbol. The delta fluorescence values were obtained by subtracting thebackground fluorescence of HEPES buffered AO at 2 μM in the absence ofDNA. The association constant for the interaction of AO with DNA wasdetermined to be 31,990±3,190 M⁻¹. As also illustrated in FIG. 3-8, theaddition of 150 μM NaCl to the initial 2 μM AO solution had little ifany effect on the binding constant between AO and the dsDNA resulting ina value of 31,690±4,980 M⁻¹.

Experiments were conducted to determine the effect of physiologicalconcentrations of NaCl on the fluorescence intensity of the AO-DNAcomplex. FIG. 3-9 depicts the spectrofluorometric analysis of AO atconcentrations ranging from 0.5 to 10.0 μM with 20 μM DNA in thepresence of, or in the absence of, 150 mM NaCl. Samples were excited ata wavelength of 485 nm and fluorescence emission values were read atλ_(em) 530 nm and λ_(em) 635 nm. Error bars shown denote standarddeviation unless hidden by legend symbols (n=4). Spectrofluorometricanalyses of 20 μM DNA at AO concentrations ranging from 0.5 to 3.0 μMrevealed a slight, but significant, increase in fluorescence values atλ_(em) 530 nm over this concentration range of AO. The addition of NaCl[150 μM] to the environment containing AO and DNA, did not contributesignificantly to the fluorescence values at either λ_(em) 530 nm orλ_(em) 635 nm until AO concentrations exceeded 2 μM as determined byANOVA statistics. At AO concentrations above 3.5 μM, the fluorescencevalues of the DNA solution at λ_(em) 530 nm in the absence of, and inthe presence of, NaCl increased 4 and 5-fold, respectfully. In theabsence of NaCl, the plot of fluorescence values at λ_(em) 530 runplateaus at AO concentrations ranging from 4.0 to 7.0 μM. Also, the datain FIG. 3-9 reveals that the fluorescence values in the presence of 150mM NaCl over the AO concentration range 4.0 to 7.0 μM, differedsignificantly (were increased) from the values obtained at λ_(em) 530 nmin the absence of NaCl. At λ_(em) 635 nm, fluorescence values increasedslightly between AO concentrations of 3.5 to 4.0 μM and continued togradually rise with increasing concentration of AO. Unlike the resultsobtained at λ_(em) 530 nm, the presence of, or the absence of, NaCl hadno significant effect on the fluorescence at λ_(em) 635 nm.

The addition of salt could possibly affect the K_(assoc) values of AOand the other reactants by modifying the charge on the reactants and/ordecreasing the influence of water molecules. To minimize AOself-association, i.e. dimerization, a relatively low concentration ofAO [2 μM] may be used. The interaction between reactants was analyzed byspectrofluorometric methods at λ_(ex) 485 nm and monitored at λ_(em) 530nm (green).

Solutions of 2 μM AO alone and in various combinations with 150 mM NaCl,5 mM CAF, 2 mM DNA were studied. Table 3-2 summarizes the results andthe statistical analysis for differences between means. TABLE 3-2 Theeffect of 150 mM NaCl, 5 mM caffeine, and/or 2 mM dsDNA on thefluorescence of a 2 μM acridine orange solution. Reactants Without NaClWith NaCl [150 mM] (2 μM AO) Mean ± S.E. (SNK) Mean ± S.E. (SNK) AO*514.0 ± 6.5 (A) 635.5 ± 17.7 (B) AO + CAF 661.0 ± 19.3 (B) 701.3 ± 53.7(B) AO + DNA 988.0 ± 11.1 (E) 795.0 ± 17.7 (C) AO + DNA + CAF 880.3 ±27.6 (D) 793.7 ± 14.4 (C)

In Table 3-2, fluorescence (λ_(ex)=485 nm and λ_(em)=530 nm) wasmeasured for AO [2 μM] in the absence of, and in the presence of, NaCl[150 mM], caffeine [5 mM), and DNA [2 mM] alone or in combination.Significant differences were found by one-way ANOVA (p≦0.05). TheStudent-Newman-Keuls (SNK) multiple range test revealed a significantdifference between means (p≦0.05), as indicated in the table.Statistical means having a different letter designation arestatistically different (p<0.05) while those means having the sameletter are not significantly different (p>0.05). It is noted that n=3unless denoted by an * which indicates n=4.

The results, as reported in Table 3-2, fell into five distinct groups,each designated by a different capitalized letter, with statisticallydifferent means (p<0.05). The lowest mean fluorescence value of 514.0was produced by 2 μM AO alone. Addition of 150 mM NaCl to 2 μM AO causeda significant increase in the mean fluorescence value from 514.0 to635.5. This value of 635.5 was not significantly different from eitherthe mean fluorescence value of 661.0 determined for AO in the presenceof 5 mM CAF or the mean fluorescence value of 701.3 determined for AO inthe presence of both 5 mM CAF and 150 mM NaCl. The addition of 2 mM DNAto the 2 μM AO solution caused the highest mean fluorescence value of988.0, which was almost double the mean fluorescence value of 514.0determined with AO alone. Yet, this value of 988.0 was significantlyhigher than the value of 795.0 obtained with the addition of 150 mM NaClto the AO-DNA solution.

The fluorescence of 2 μM AO at 8_(em) 530 nm was increased 92% by theaddition of 2 mM DNA and by 29% by the addition of 5 mM CAF. Thepresence of 150 mM NaCl further increased the fluorescence intensity ofAO in the presence of CAF. The addition of NaCl significantly reducedthe ability of DNA to increase the fluorescence intensity of AO.Therefore, the addition of either DNA or CAF when in the presence of 150mM NaCl resulted in an increase in fluorescence intensity of AO by 54%and 36%, respectively.

The results from the previously described experiments characterize thespecific interactions of AO to each of the binding species, CAF or dsDNAindividually, while keeping the concentration of AO fixed at 2μM.

Spectrofluorometric analyses at λ_(ex) 485 nm and λ_(em) 530 nm wereperformed on solutions of 2:M AO in combination with 5 mM CAF and 2 mMdsDNA, with and without 150 mM NaCl. Statistical analysis of theseresults are also reported in Table 3-2. Means with different letterdesignations are statistically different (p<0.05) while means containingthe same letter designation had means that were not significantlydifferent. Manipulation of the AO-DNA solution by the addition of 5 mMCAF significantly lowered (p<0.05) the mean fluorescence value from988.0 to 880.3. Yet, this value of 880.3 was significantly higher thanthe value of 795.0 obtained with the addition of only 150 mM NaCl to thesame AO-DNA solution. Note that CAF had no significant effect on thefluorescence of the AO solution when in the presence of NaCl (SNK letterdesignation “B”). The combination of 2 mM DNA, 5 mM CAF, and 150 mM NaClwith AO gave a mean fluorescence value of 793.7 that was notsignificantly different from the mean value of 795.0, obtained for AO inthe presence of 2 mM dsDNA plus 150 mM NaCl but in the absence of CAF.

Bound AO is known to fluoresce at two distinct wavelengths based on thetype of molecular association that occurs. When AO bindselectrostatically to a compound (e.g. RNA), it has a characteristic redfluorescence with λ_(max) emission of 635 nm. Alternatively, when AOintercalates, as occurs with DNA, it fluoresces green at a λ_(max) of530 nm (Haugland, 1994). The fluorescence of free unbound AO monomer isweak at low concentrations (1-10 μM and, therefore, contributes littleto background fluorescence. The electrostatic interaction of AO withitself can, therefore, be monitored at λ_(em) of 635 nm while theformation of a stacked dimer can be followed by monitoring thefluorescent emission at 530 nm.

The increase of NaCl concentration to 150 mM resulted in an increase inthe optical absorbance at 268 nm in relationship to the absorbance at491 nm indicative of an increase in the concentration of dimer insolution. Also, the addition of 150 mM NaCl resulted in an increase inK_(assoc) from 37,410 M⁻¹ to 118,000 M⁻¹ at AO concentrations rangingfrom 0.5 μM to 5.0 μM.

In summary, the formation of the dimer or stacked AO aggregate isdependent, in part, upon the concentration of AO. The addition of saltto AO solutions facilitates dimer formation at lower AO concentrationsbut has minimal additional effects at AO concentrations above 5 μM. Thesharp increase in green fluorescence (λ_(em) 530 nm) at an AOconcentration of 5 μM is probably due to a shift in AO equilibriumtoward the formation of AO dimers. The critical concentration for thisphenomenon seems to be approximately 5-6 μM. Below this concentration,AO exists primarily as a monomer with an increased formation of itsdimer species as the concentration of AO increases toward 5 μM. Atconcentrations above 5 μM, molecular AO exists in solution as a monomer,a dimer, and possibly as unstacked aggregates of dimers. Fluorescenceanalysis allowed for the clear distinction and specific monitoring ofthe dimeric and monomeric forms of the AO molecule.

The optical titration and spectral analysis studies of CAF with theDNA-intercalator AO demonstrates that CAF can complex with AO via a B-Btype interaction. An important force in the formation of such complexesappears to be van der Waals interactions resulting in maximal ringoverlap between the two molecules of the complex. Complexation betweenCAF and aliphatic mutagens show much lower binding energies relative toCAF complexes with aromatic intercalators. The corresponding bindingconstant for CAF-AO complexes is on the order of 250 M⁻¹ (Table 3-3). InTable 3-3, absorbance was scanned between 400 nm and 550 nm.Fluorescence excitation was at 485 nm whereas emission was observed at530 nm. TABLE 3-3 Summary of association constants (K_(assoc)) foracridine orange (AO) with itself, with caffeine (CAF), and with dsDNA inthe absence of and in the presence of 150 mM NaCl. Complex Method K ±S.D.[M⁻¹] Concentration Source AO-AO Absorbance^(a) 29,100 AO [20 μM]Kapuscinski & plus NaCl^(b) 42,500 Kimmel, 1993 Absorbance 46,840 ±5,967 AO [2.5-20 μM] This study plus NaCl 47,510 ± 2,420 AO [10-45 μM] ″Absorbance 49,530 ± 2,953 AO [10-80 μM] This study Fluorescence 37,410 ±4,758 AO [0.5-5.0 μM] This study plus NaCl 118,000 ± 16,120 AO [0.5-5.0μM] ″ Fluorescence 46,700 ± 1,807 AO [6.0-10.0 μM] This study plus NaCl41,820 ± 3,671 AO [6.0-10.0 μM] ″ AO-CAF Absorbance 256 ± 5  AO [10 μM];CAF [1-5000 μM] This study Absorbance 258 ± 5  AO [10 μM]; CAF [1-5000μM] Larsen et al., 1996 Absorbance 258 ± 7  AO [20 μM]; CAF [1-2000 μM]Kapuscinski & plus NaCl^(b) 169 ± 9  AO [20 μM]; CAF [1-2000 μM] Kimmel,1993 Fluorescence 254 ± 27 AO [2 μM]; CAF [1-20 μM] This study plus NaCl216 ± 20 AO [2 μM]; CAF [1-20 μM] ″ AO-DNA Absorbance 33,510 ± 285   AO[0.1-3 μM]; DNA [100 μM] This study Absorbance 31,960 ± 316   AO[0.8-3.4 μM]; DNA [20 μM] ″ Absorbance 37,660 ± 431   AO [3 μM]; DNA[5-170 μM] ″ Fluorescence 31,990 ± 3,190 AO [2 μM]; DNA [>20 μM] ″ plusNaCl 31,690 ± 4,890 AO [2 μM]; DNA [>20 μM] ″^(a)K_(assoc) calculated using absorbance data and a mathematical model.^(b)NaCl concentration of 150 mM

The spectrophotometric analysis of the AO-DNA binding reported hereinsuggests the presence of a single bound species at high DNA to AO ratiosand, therefore, only one mode of binding (a single isobestic point couldbe easily identified during the spectrophotometric analysis only at DNAto AO molecular ratios above 5.0). The measurement of an associationconstant or ‘affinity’ for the AO-DNA system, under such conditions, wasassessed by both absorbance studies and fluorescence studies and theK_(assoc) values were found to be similar (Table 3-3).

Data from optical titrations suggest that the AO-AO self-stacking ordimer formation is practically eliminated at high DNA to AO ratios.These data suggest that the binding process for AO to DNA at lowermolecular ratios can be divided into two different processes as has beendescribed for most of the acridine derivatives: one of high affinity(type I) and one of lower affinity (type II). The type I process isstrong and corresponds to the intercalation of the AO into the DNAdouble helix at low binding ratios. Type II process is weak and isnormally attributed to an external electrostatic binding at high bindingratios. The use of different fluorescence emission wavelengths allowedfor the calculation of association constants for each kind of bindinginvolved in the interaction process. Therefore, binding valuesdetermined from data obtained at λ_(em) 530 nm should be considered asrelative to the intercalation or B-B binding (stacking) of AO to DNA.The K_(assoc) values obtained at low AO to DNA ratios (intercalationprocess) suggest that the fluorometric determination at λ_(em) 530 nmonly allows the calculation for the type I strong binding modeassociation constant.

At low DNA to AO molecular ratios, a decrease in the fluorescenceintensity of AO was observed when interacting with DNA. AO has a strongbinding affinity for DNA, especially at low AO concentrations. Thisbehavior is sensitive to the concentration of the acridine chromophoreas stronger binding between AO and DNA is seen at lower ratios of AO toDNA base pairs (AO concentrations <3 μM).

An increase of NaCl concentration to 150 mM resulted in an increase inK_(assoc) for the dimerization of AO from 37,410 M⁻¹ to 118,000 M⁻¹ atAO concentrations ranging from 0.5:M to 5.0 :M (inset to FIG. 3-2). Thisis likely attributed to the stabilization of the AO-AO dimer bydecreasing the contribution of water molecules to the solvation of theAO monomer by competing with the electrostatic charge due to theprotonization of the nitrogen on AO. Thus the higher K_(assoc) value inthe presence of 150 mM NaCl is expected to shift the equilibrium betweenmonomeric AO and dimeric AO to favor the formation of AO dimers. Even atrelatively low AO concentrations [2 μM], physiological concentrations ofNaCl caused a 23% increase in fluorescence values at λ_(em) 530 nm,indicative of an increase in dimer formation (see Table 3-2).

The solution containing AO and DNA gave a fluorescence value almostdouble that of AO alone (Table 3-2) reflecting the high degree to whichAO intercalated into the DNA. The addition of 150 mM of NaCl to asolution containing the AO and DNA caused a 20% reduction in thefluorescence of the solutions In solutions containing NaCl, an increasein the ionic strength causes DNA to lose some of its ability to formelectrostatic bonds with AO. At a molecular ratio of 1000:1 (2 mM DNA: 2μM AO), this should have little if any effect on the fluorescenceintensity of the AO-DNA complex. The addition of NaCl causes a shift inthe amount of free AO monomer available for intercalation into DNA byincreasing the concentration of AO-AO dimer. This increase in theformation of AO-AO dimers would cause a decrease in fluorescenceintensity by reducing the concentration of AO-DNA complexes formed by aratio of 2 to 1 for every dimer formed.

In a solution containing all three reactants, the equilibrium in theAO-CAF-DNA system is controlled by four parameters, i.e., K_(AO-AO),K_(AO-CAF), K_(AO-DNA (i)), and K_(AO-DNA (e)) which represent (at AO>5μM) the association constants for AO-AO, AO-CAF, AO-DNA (intercalation),and AO-DNA (electrostatic) respectfully. The K_(eq) for the system wouldbe:K _(eq) =K _(AO-AO) +K _(AO-CAF) +K _(AO-DNA (i)) +K _(AO-DNA (e))

At any given concentration of AO, at constant temperature, pH, andpressure, K_(eq) is a constant. Changes in the K_(assoc) values forAO-AO, AO-CAF, or AO-DNA_((i/e)) will not affect the overall K_(eq) ofthe system, but will instead affect the K_(assoc) values between theother reactants. Results from the interaction of all three reactants inthe absence of and in the presence of NaCl, support this concept.However, the K_(assoc) values for each reactant reflects the molecularinteraction with the AO monomer and assumes that the concentration of AOin solution is entirely in monomeric form. At AO concentrations <5 μM,this is a reasonable assumption and the interpretation of fluorometricmeasurement of the interactions between AO, dsDNA, CAF, and NaCl arerelatively simple. At AO concentrations >5 μM, the assumption that AO isexclusively in monomeric form is invalid. Even at AO concentrations >5μM, one can still interpret the spectrofluorometric data at λ_(em) 530nm as discussed next.

The model presented in FIG. 3-10 illustrates the molecular populationsof AO at 2 μM and at 20 μM. At a concentration of 2 μM, AO is almostexclusively in monomeric form whereas at a concentration of 20 μM, thereexists a dynamic equilibrium between the monomeric and dimeric forms.The monomeric form of AO reacts with CAF to form an AO-CAF complex (alsoshown in FIG. 3-10) and only the monomeric form of AO can intercalateinto dsDNA. The availability of monomeric AO is critical to itsintercalation into dsDNA and/or its complexation with CAF. Conditionsthat affect the equilibrium between the dimeric and monomeric forms ofAO, such as NaCl, effect the overall concentration and availability ofmonomeric AO. Table 3-2 shows that the addition of CAF to the AO-DNAsystem causes a significant decrease in fluorescence with no significantdecrease upon the addition of NaCl. This suggests that the relativelyhigh concentration of CAF (5 mM) is able to reduce the amount of AOintercalated into DNA through competitive complexation of monomeric AO,based on the laws of mass action. The addition of CAF to the AO-DNA-NaClsystem causes no significant change in fluorescence, because CAF is in anon-charged state and because the K_(assoc) value for the AO-DNA complexis much greater than that for AO-CAF and, therefore, is the primaryforce controlling the equilibrium and availability of monomeric AO.

The results of these experiments reveal that the interaction of CAF withincreasing AO concentration follows the law of mass action in thatfluorescence increases proportional to the increase in AO concentration.The addition of dsDNA to the AO-CAF system causes a significantreduction in fluorescence response. This is attributed to theelectrostatic bonding of AO to the DNA backbone and, therefore, areduction in available AO monomers for AO-AO dimerization and forcomplexation with CAF thus resulting in the lowering of the fluorescenceintensity. The addition of NaCl to this system slightly increases theamount of fluorescence along the series of increasing AO concentrationsand can be attributed to charge neutralization of the phosphate group onthe DNA backbone freeing up monomeric AO as well as the ability of NaClto stabilize AO-AO dimers. The charge neutralization of the phosphatebackbone on DNA also results in an equilibrium shift to more availablemonomeric AO thus contributing to the formation of AO-CAF complexesand/or AO-DNA intercalation that results in a higher fluorescentintensity.

The use of xanthines is not limited to merely binding to free DNAintercalators. Xanthines can also be used as interceptors of compoundsintercalated into DNA. As discussed previously, the law of mass actioncan be exploited to inhibit the binding of DNA intercalators. In asimilar fashion, because the DNA intercalators are in dynamicequilibrium with polynucleotides, the law of mass action can beexploited to remove bound intercalators. Furthermore, the use of theinterceptor molecule is not limited to modified xanthines. Any moleculethat binds with the DNA intercalator through the law of mass action canbe used. Examples include, but are not limited to, purines, pyrimidines,uric acid, porphyrin, polynucleotides, DNA, RNA, PNA, and combinationsthereof.

EXAMPLE 4

In another implementation of the invention, the cationic fluorescent dyeacridine orange (AO; 3,6-bis[dimethylamino]acridine hydrochloride, >99%purity), was obtained from Aldrich Chemical Company, Milwaukee, Wis.,and HEPES buffer was obtained from Mediatech, Inc., Herndon, Va. Highlypolymerized herring sperm deoxyribonucleic acid sodium salt was obtainedfrom ACR_S Organics, Pittsburgh, Pa. was used without furtherpurification. Caffeine (CAF; 1,3,7-trimethyl xanthine, Aldrich), andthree other methylated xanthines, 1,7-dimethyl xanthine, theophylline(3,7-dimethyl xanthine, ACR_S) 1,3,7,9-tetramethyl-8-oxy xanthine (TMU;tetramethyl uric acid, ICN Biomedicals, Inc., Aurora, Ohio),aminophylline (theophylline ethylenediamine) were also obtainedcommercially at 99% purity and used without further purification. Stocksolutions of CAF and AO were prepared at a concentration of 50 mM. Astock solution of herring sperm DNA was prepared fresh the day of use.The concentration for dsDNA was based on the average molecular weight ofall four nucleoside bases to obtain an approximate formula weight of 340g/mole (one base) or 680 g/mole (one base pair or “bp”). This methodassumed that each base occurs equally within the DNA. Using the formulaweight of 340 g/mole, stock solutions of DNA were prepared at aconcentration of 5 mM. All solutions and subsequent dilutions wereprepared by dissolving the appropriate weighed amount in 5.0 mM HEPESthat was adjusted to pH 7.0 with 0.1 M NaOH. AO was prepared in a stocksolution at a concentration of 50 mmol in 5 mmol HEPES buffer (1.0 Msolution, Mediatech, Inc., Herndon, Va.). AO was chosen as arepresentative DNA intercalator because of its known spectralcharacteristics and its wide use as a fluorescence chromophore markerfor both DNA and RNA (Robinson et al., 1973; Kapuscinski, 1990;Kapuscinski and Darzynkiewicz, 1987, 1990; von Tscharner and Schwarz,1979). Specifically, AO exhibits two λ_(max) fluorescence peaks,depending upon the type of interaction it has with itself or othercompounds (Larsen et al., 1995; Haugland, 1994; Darzynkiewicz et al.,1996). For example, when AO intercalates into DNA, presumably by forminga B-B interaction or stacking complex, it has a characteristic greenfluorescence maximum at about λ_(em) 526 nm (green). Alternatively, whenAO associates with RNA or other compounds electrostatically (ionic)through charge transfer it has a characteristic fluorescence maximum atabout λ_(em) 650 nm (red). The two emission wavelengths used in thisstudy, λ_(em) 635 nm and λ_(em) 530 nm, represent the approximateλ_(max) for the fluorescence emission of AO when electrostaticallyassociated with other compounds (635 nm) and in a stacked (B-B)association with itself or another compound or compounds (530 nm).

All stock solutions and subsequent dilutions were prepared by dissolvingthe appropriate weighted amount of compound in 5.0 mmol HEPES adjustedto pH 7.0 with 0.1 M NaOH.

EXAMPLE 5

In another inplementation of the invention, depurinated nucleotides canbe used as sites that only minimally bind the intercalator acridineorange.

Chicken erythrocytes were chosen as one implementation of thisinvention. These nucleated cells have mature erythrocytes with no RNA.Blood was taken from a mature chicken. The blood was drawn from a wingvein into 50 ml glass heparin containing tubes to prevent clotting. Thisblood was used to make smears on microscope slides. These blood smearedslides were allowed to dry at room temperature and were stored inmicroscope boxes.

Depurination of dsDNA within the nuclear chromatin of chickenerythrocytes was accomplished by using a modification of the Feulgenmethod (Feulgen and Rossenbeck, 1924). Specifically, the nucleatedchicken erythrocyte slide preparations were rinsed in a 1 N solution ofHCl at room temperature for 60 seconds. This was followed by placementof the slide in a pre-heated (55° C.) solution of 1 N HCl for 10 minutesunder static conditions followed by several rinses in distilled water atroom temperature. Slides were allowed to dry at room temperature thenstained with AO at two different concentrations, one at 5 mM and theother at 10 μM for 15 min and 2 min, respectively. The depurination ofDNA was expected to remove the site on the DNA polymer where AOintercalation can occur.

Solutions of AO were prepared at 5 mM and 10 μM concentrations indistilled water. Microscope slides smeared with chicken erythrocyteswere stained by placing the slide into a glass slide holder containing50 ml of AO solution under static conditions from 2 min to 30 min,followed by three 10 second rinses in distilled water before drying atroom temperature. The staining of nuclear chromatin dsDNA of the chickenerythrocyte at high AO to DNA ratios allowed for both the intercalationof AO into dsDNA and for AO to electrostatically bind to the phosphatebackbone of the nuclear chromatin dsDNA strand. Alternatively, thestaining at low AO to DNA ratios allowed for only intercalation into thedsDNA. Control slides consisted of chicken erythrocyte smeared slidesthat were subsequently placed in pure distilled water (no stain) priorto drying (negative control) and blank slides (no erythrocytes) placedin AO staining solutions then dried. Processed slides were stored in thedark then read and photomicrographs taken (Zeiss Model 63 Microscope,1600 ASA Kodak film) soon after removal from the dark to avoidphotobleaching.

Table 4-1 lists the results observed upon staining chicken erythrocytenuclear chromatin with two different concentrations of AO before andafter acid hydrolysis to produce apurinic acid (ap). Results from theseexperiments show that when the purine bases are removed from dsDNAwithin the nuclear chromatin, AO is unlikely to form a complex asevidenced by the lack of green fluorescence at λ_(em) 530 nm. However,electrostatic binding continues to occur as evidenced by the continuedpresence of red fluorescence at λ_(em) 635 nm. TABLE 4-1 Color observedupon staining chicken erythrocyte nuclear chromatin with acridine orangebefore and after acid hydrolysis of dsDNA to apurinic acid (ap) asdetermined by fluorescence microscopy. Fluorescent Color ChickenErythrocytes 10 μM 5 mM Chromatin Green Red apChromatin No Color Red

In the example shown in Table 4-1, the chicken erythrocytes were smearedand dried on a microscope slide prior to exposure to two differentconcentrations of AO (10 μM and 5 mM), rinsed with distilled water, andallowed to dry. Erythrocytes were examined with fluorescence microscopyand the color of the nuclei recorded. Xanthine exposure times for 10 μmAO and 5 mM AO were 15 min and 2 min, respectively. The depurinatednucleotides (apChromatin) exhibited essententially no green color in thepresence of 10 μM AO illustrating essentially no binding thereto.

EXAMPLE 6

In another implementation of the invention, modified xanthines are usedto inhibit the binding of DNA intercalators. By providing an excess ofxanthines as compared to DNA intercalators, the binding of theseintercalators can be inhibited for more than 10 minutes.

Microscope slides coated with chicken erythrocyte were stained with 10μM AO at three separate areas on the microscope slide as describedabove. Slides were exposed to five different xanthies at four differentmolar ratios for three different periods of time. The xanthines,theophylline, caffeine, 1,3,7,9-tetramethyl uric acid, andaminophylline, were prepared with 10 μM AO at molar ratios of 50:1,500:1, 2500:1, 5000:1 xanthine to AO in distilled water. Microscopeslides coated with chicken erythrocytes were exposed to a mixture ofxanthine/AO at each molar ratio for periods of 10 min, 1 min, and 10seconds under static (non-agitated) conditions. Slides were also treatedwith mixtures of dsDNA/AO solutions under the same conditions to studythe effect of a competitor DNA molecule with a significantly higherK_(assoc) value for AO than the xanthines. Following exposure to thexanthine/AO or to the DNA/AO solutions, the slides were rinsed indistilled water, allowed to dry at room temperature, examined byfluorescence microscopy, and the color of the nuclei recorded. Resultswere recorded as follows: 0=no blocking effect; 1=slight blocking;2=moderate blocking; 3=almost total blocking (very slight greenfluorescence detectable); 4=complete blockage (no green fluorescencedetectable).

Experiments testing the ability of different xanthines with knownbinding affinities for AO, as well as pure dsDNA, to inhibit theintercalation of AO into chicken erythrocytes chromatin were performed(results summarized in Table 4-2). At AO concentration of 10 μM,xanthines with the highest binding affinity for AO tended to blockintercalation relative to the strength of their binding affinity andmolar ratio to AO. For example, aminophylline with a K_(assoc) value of596.2 M⁻¹ showed a significant ability to block AO intercalation and tomaintain that inhibition even at 10 minutes exposure. The efficacy ofblocking was correlated to the molar ratio of aminophylline to AO. Asthe molar ratio of aminophylline to AO increased by two orders ofmagnitude from 50:1 to 5000:1 the efficacy of inhibition also increasedproportionally. Theophylline, with a binding affinity of 160.8 M⁻¹ hadminimal effect even at an exposure time of 10 seconds. Exposure to dsDNAwith the highest binding affinity to AO (˜4000 M⁻¹) was more effectivethan any of the xanthines tested at blocking AO from staining nuclearchromatin. TABLE 4-2 Effect of increasing exposure time and xanthineconcentration on the ability of xanthines with increasing K_(assoc)values for acridine orange to block acridine orange intercalation intochicken erythrocyte nuclear chromatin as determined by fluorescencemicroscopy. Compound Xanthine/AO Exposure Time (K_(assoc) M⁻¹)* MolarRatio 10 min 1 min 10 sec Theophylline 5000:1 0 0 0 (157) 2500:1 0 0 0 500:1 0 0 0  50:1 0 0 0 Caffeine 5000:1 1 2 3 (256) 2500:1 0 1 2  500:10 0 2  50:1 0 0 0 1,3,7,9-Tetramethyl Uric Acid 5000:1 2 2 2 (552)2500:1 1 1 0  500:1 0 0 0  50:1 0 0 0 Aminophylline 5000:1 3 3 3 (596)2500:1 1 2 3  500:1 0 2 2  50:1 0 0 0 DNA  500:1 2 3 3 (˜4000)  50:1 1 23   5:1 0 0 0   1:1 0 0 0

In the implementation shown in Table 4-2, the relative intensity of thegreen fluorescent color (λ_(em) 360 nm) utilizing a red blocking filter(≧600 nm) was used as the indicator of the intensity of staining. Themolar ratio is expressed as the ratio of xanthine concentration to 10 μMAO. Results were recorded as follows: 0=no blocking effect; 1=slightblocking; 2=moderate blocking; 3=almost total blocking (very slightgreen fluorescence detectable); 4=complete blockage (no greenfluorescence detectable). *K_(assoc) values for the xanthines are takenfrom Table 2-1 and the K_(assoc) values for DNA were taken from Table3-2.

The application of this invention is not limited to the modifiedxanthines tested or to the concentrations used. This invention isapplicable to a wide range of modified xanthines, including, but limitedto 1-methyl xanthine, 1,3-dimethyl xanthine, 3,7-dimethyl xanthine,1,7-dimethyl xanthine, 1,3,7-tri-methyl xanthine, 1,3-dimethyl xanthine,1,3-dimethyl-8-oxy xanthine, 1,3-dimethyl-8-chloro xanthine, and1,3,7,9-tetramethyl-8-oxy xanthine. Additionally, the excess amount ofxanthines need not be limited to that given in Table 4-2. Variations ofXanthine to intercalator ratio are included within the scope of thisinvention. In addition, an polynucleotide, whether it is DNA, RNA, orPNA may be used in either a single, double, or higher order strandedstructure. Furthermore, the use of the interceptor molecule is notlimited to modified xanthines. Any molecule that binds with the DNAintercalator through the law of mass action can be used. Examplesinclude, but are not limited to, purines, pyrimidines, uric acid,porphyrin, polynucleotides, DNA, RNA, PNA, and combinations thereof. Inaddition, this invention also includes the varying the saltconcentration as discussed previously to stabilize the dimer fomation ofintercalator molecules.

EXAMPLE 7

In still another implementation, the modified xanthine may be used tointercept and remove the an intercalator bound to a DNA complex. Forthis aspect of the invention, an excess amount of modified xanthines aredelivered to the DNA/intercalator complex. Because intercalatormolecules are in dynamic equilibrium with the DNA, an excess of modifiedxanthine can be used to remove the intercalator molecule fromDNA/intercalator complex.

Experiments were conducted to determine whether xanthines with knownbinding affinities to AO could compete with nuclear DNA for binding toAO and, therefore, be able to remove AO from its intercalation positionwithin nuclear chromatin. Fluorescence microscopy was used to observethe fluorescent color produced by the AO-DNA complex following thestaining of chicken erythrocyte nuclear chromatin with AO at both 5 μMand 5 mM. A red fluorescence was observed at higher AO:bp ratios whenthe amount of AO far exceeds the number of possible intercalation sites.This red fluorescence, is attributed to its electrostatic bonding to thecharge groups on the phosphate backbone of the DNA molecule and, fullymasks the green fluorescence of the intercalated AO. Alternatively, atlow AO:bp ratios when the amount of AO is less than or equal to thenumber of available intercalation sites, green fluorescencepredominantly occurred, characteristic of AO intercalation betweenpurine base pairs on dsDNA (Haugland, 1994). Since the binding affinityfor AO intercalation (green fluorescence) is significantly higher thanits affinity to form electrostatic bonds (red fluorescence), AO prefersto bind to dsDNA via intercalation, FIG. 4-2 shows two photomicrographstaken of microscope slides smeared with chicken erythrocytes thenstained with 10 mM AO at two exposure times to illustrate thedifferential fluorescence color of nuclear chromatin. Nuclei (top)exposed to the AO solution for 10 minutes then rinsed with waterfluoresce red (electrostatic bonding) while nuclei (bottom) exposed toAO for 10 seconds then rinsed with water, fluoresce green(intercalation). A rinse of the slides with 50 mM of CAF for 10 minutesreduced the fluorescence intensity to background level.

Table 4-3 lists the results from the rinsing of chicken erythrocyteswith different xanthines having different binding affinities for AO. Thetwo xanthines with the lowest K_(assoc) values had little significanteffect on the observed fluorescence produced by the AO stained nuclearchromatin However, as the K_(assoc) value of the xanthine increased to˜256 M⁻¹, as seen with CAP, reversibility of AO staining to dsDNAoccurred as evidenced by the disappearance of both, green fluorescencein erythrocytes lightly stained with AO (low AO:bp ratio) and, a slightbut observable reduction in red fluorescence for erythrocytes highlystained with AO (high AO:bp ratio). Rinses with both,1,3,7,9-tetramethyl uric acid (K_(assoc) 552 M⁻¹) and aminophylline(K_(assoc) 596 M⁻¹), resulted in a removal of most, if not all, greenand red color as observed under fluorescence microscopy. These resultsindicate that xanthines with higher K_(assoc) values are more effectivein the removal of AO from chicken erythrocyte nuclear chromatin. TABLE4-3 Nuclear color observed by fluorescence microscopy after stainingchicken erythrocyte nuclear chromatin with acridine orange at either 5μM or 5 mM, before and after xanthine rinses, and after re-staining withacridine orange. COMPOUNDS AO [5 μM] AO [5 mM] K_(assoc) After AfterXanthine Rinse mM (M⁻¹) Stain Rinse Re-stain Stain Rinse Re-stainTheophylline 50 161 Green Green Green Red Red Red Caffeine 50 256 GreenNone Green Red Red- Red Green* Tetramethyl uric acid 50 552 Green NoneGreen Red None Red Aminophylline 50 596 Green None Green Red None Red*Transitional from red to green fluorescence having a color thatappeared greenish yellow.

The invention is not limited to chicken erthrocyte DNA. The applicationof this invention is not limited to the modified xanthines tested or tothe concentrations used This invention is applicable to a wide range ofmodified xanthines, including, but limited to 1-methyl xanthine,1,3-dimethyl xanthine, 3,7-dimethyl xanthine, 1,7-dimethyl xanthine,1,3,7-tri-methyl xanthine, 1,3-dimethyl xanthine, 1,3-dimethyl-8-oxyxanthine, 1,3-dimethyl-8-chloro xanthine, and 1,3,7,9-tetramethyl-8-oxyxanthine. Additionally, the excess amount of xanthines need not belimited to that given in Table 4-2. Variations of Xanthine tointercalator ratio are included within the scope of this invention. Inaddition, an polynucleotide, whether it is DNA, RNA, or PNA may be usedin either a single, double, or higher order stranded structure.Furthermore, the use of the interceptor molecule is not limited tomodified xanthines. Any molecule that binds with the DNA intercalatorthrough the law of mass action can be used. Examples include, but arenot limited to, purines, pyrimidines, uric acid, porphyrin,polynucleotides, DNA, RNA, PNA, and combinations thereof. In addition,this invention also includes the varying the salt concentration asdiscussed previously to stabilize the dimer fomation of intercalatormolecules.

EXAMPLE 8

This invention can also be applied to staining and/or labeling of DNA.Chicken erythrocytes were used as example system to illustrate the useof labeling of DNA.

Microscope slides smeared with chicken erythrocytes were stained witheither 5 μM or 5 mM AO for a period of 30 minutes followed by three 10second rinses in distilled water before drying at room temperature.Slides were then examined by fluorescence microscopy using a, ZeissModel 63 Microscope with a xenon source fluorescent emitter. In someinstances, a blocking filter was used to filter out fluorescenceemission in the red and higher wavelengths. Photomicrographs were takenwith Kodak Gold 35 mm ASA 1600 speed film, developed and color printsmade. Comparative photomicrographs were taken at the same magnificationand at the same exposure time. Selected photomicrographs were digitallyscanned (Hewitt-Packard, Model 4L Scanner, Palo Alto, Calif.) and storedon 100 MB ZIP cartridges.

Slides were then exposed to 50 ml solutions of theophylline (50 mM),caffeine (50 mM), 1,3,7,9-tetramethyl uric acid (50 mM), andaminophylline (50 mM) for 1 hour under static conditions, rinsed withdistilled water, then, allowed to dry at room temperature. Slides wereagain examined by fluorescence microscopy and fluorescence color of thenuclei recorded. Slides were again stained with 5 mM AO then re-examinedby fluorescence microscopy and fluorescence color of the nucleirecorded.

In the example shown in Table 4-3, the chicken erythrocytes were smearedand dried on a microscope slide prior to exposure to the AO solution for30 minutes followed by water rinses to remove excess AO. Followingdrying, each slide was observed under fluorescence microscopy thenplaced in one of the xanthine solutions (50 ml volume) for 1 hour understatic conditions, rinsed with water, then dried prior to being observedby fluorescence microscopy without a red blocking filter. Finally, eachslide was restained following the initial staining procedures and againobserved by fluorescence microscopy. K_(assoc) values are taken fromTable 2-1 and are expressed in M⁻¹.

To demonstrate that dsDNA would give the same fluorescent color resultsas chicken erythrocyte nuclear chromatin, dsDNA in distilled water wasplaced on microscope slides and was allowed to dry. Additionally thisinvention can be used to not only inhibit the binding of theintercalator, but to actually remove the intercalator from the DNA usingthe law of mass action.

In another implementation, giant polytene chromosomes were prepared bythe squash technique from the salivary gland of Drosophila melagasterlarva The salivary gland was placed on a microscope slide, squashed witha glass cover slide, fixed with acidic acid, rinsed with distilledwater, and allowed to dry (kindly provided by Linda Chadwell,UTHSCSA-IBT). Three known DNA intercalators, ethidium bromide (98%purity, Aldrich Chemical Company, Milwaukee, Wis.), doxorubicin (98%,Sigma Chemnical Company, St. Louis, Mo.), and acridine orange plus theminor groove binding DNA stain Hoechst 33258 (98%, ACROS ChemicalCompany, NJ) were prepared in distilled water at a concentration of 1 mM(for chemical structures see FIG. 4-1, where acridine orange (3,6-bis[dimethylamino] acridine) is shown in Panel A, ethidium bromide is shownin Panel B, doxorubicin is shown in Panel C, Hoechst 33258 is shown inPanel D). The slides with prepared chromosomes were immersed in thesestains for a period of 30 minutes, rinsed with distilled water, anddried at room temperature. Fluorescence microscopic analysis wasconducted at λ_(ex) 360 nm and photomicrographs were conducted on aZeiss Model 63 Microscope with a xenon source fluorescent emitter.Photomicrographs were taken using Kodak Gold 35 mm ASA 1600 speed filmnoting the position of one individual chromosome on the slide. Thestained slides were then subjected to 100 mM caffeine or 100 mMaminophylline under static conditions for a period of 15 minutes, rinsedwith distilled water, then allowed to dry at room temperature. Thepresence of the same individual unstained chromosome in the field wasconfirmed by phase contrast microscopy. The chromosome was viewed againunder fluorescence microscopy at λ_(em) 360 nm, and color recorded.Slides were restained by immersion in the 1 mM stain for a period of 30minutes, rinsed with distilled water, and dried at room temperature, andthe same individual chromosome was examined by fluorescence microscopyand the fluorescence color recorded.

Microscope slides (slides) coated with chicken erythrocytes were stainedby placing the slide into a 50 ml beaker filled with a 1 mM solution ofAO prepared in distilled water for approximately 10 seconds (static)followed by three successive 10 second rinses in distilled water beforedrying at room temperature. Care was taken to store all slides in thedark when not in use to avoid photobleaching. Stained slides were driedprior to being exposed to 50 ml of a 100 mM solution of either CAF oraminophylline (AM) for set time periods under static conditions.Exposure to the xanthine solution was followed by three 10-second rinseswith distilled water and drying of the slides in the dark prior tofluorescence analysis. Control slides consisted of erythrocyte coatedslides that were subsequently placed in only distilled water (no stain)prior to drying, blank slides (no erythrocytes) placed in AO stainingsolutions then dried served as negative controls, and erythrocyte coatedcells stained with AO but exposed to only distilled water and not axanthine solution served as positive control. The stained microscopeslides were then attached by tape to a plastic frame (upside down96-well plate cover) and fluorescence determined using a Perkin-ElmerModel HTS 7000 Bio Assay Reader with λ_(ex) 485nm and λ_(em) 530 withthe gain set at 40. Neither the blank slide exposed to AO nor theerythrocyte smeared slide exposed to distilled water resulted indetectable fluorescence.

These DNA coated slides were then treated in the same xanthine solutionwashes as the slides coated with chicken erythrocytes. Examination ofthese DNA coated slides using the same AO staining conditions andxanthine exposure conditions used for the chicken erythrocytes did yieldthe same color fluorescent microscopy changes seen in the erythrocytenuclear chromatin. Re-staining of these DNA coated slides with the AOsolution following xanthine treatments confirmed that stainable DNA wasstill present on the slides.

FIG. 4-3 depicts three photomicrographs of the same Drosophila polytenechromosome: after staining with the DNA minor groove binder Hoechst33258 dye (Panel A), after rinsing with CAF for 15 minutes (Panel B),and a phase contrast verification of the presence of the chromosomeafter the CAF rinse (Panel C). Table 4-4 summarizes the results from theexperiments using caffeine (CAF) and aminophylline (AM) to remove thefollowing DNA intercalators: doxorubicin, ethidium bromide, acridineorange, and the DNA minor groove binder Hoechst 33258 from their bindingsites within polytene chromosomes. The results demonstrate the abilityof CAF or of AM to extract intercalated AO from dsDNA polytenechromosomes. AO appeared to be completely removed with AM washes whileexposure to CAF washes did not completely remove the stain from thechromosome under these experimental conditions. Results from tests withthe other DNA intercalators show that either aminophylline or caffeinecan be used to remove these DNA intercalators from the chromosome bands.Results from tests with the DNA minor groove binder Hoechst 33258 dyedemonstrate an apparently complete removal of Hoechst 33258 from thechromosomes with the addition of either CAF or AM. Doxorubicin, known toform a binding complex with caffeine, also appeared to be completelyremoved from the chromosomes upon exposure to CAF. However, exposure toAM only moderately removed doxorubicin from the chromosome. Finally,using ethidium bromide as the DNA intercalator revealed that AM was lesseffective in removing the ethidium bromide molecule from the chromosomethan was CAF. With all DNA intercalators, the staining of chromosomebands was intense before the xanthine rinses and was again intensefollowing re-staining. TABLE 4-4 The removal of the DNA intercalatorsethidium bromide, doxorubicin, acridine orange, and the DNA minor groovebinder Hoechst 33258 from polytene chromosome bands by 15 minute washtreatment with 100 mM aminophylline or with 100 mM caffeine followed bydistilled water rinse and drying. Staining Degree of Stain RemovalRe-staining before Exposure following Xanthine to CAF Exposure toXanthine DNA Intercalator Exposure (100 mM) AM (100 mM) ExposureAcridine Orange Banding Almost Complete Complete complete BandingEthidium Banding Complete Almost Complete Bromide complete BandingDoxorubicin Banding Complete Moderate Complete Banding Hoechst 33258Banding Complete Complete Complete Banding

To verify the observations and results from the experiments usingfluorescence microscopy, experiments were conducted to quantify theremoval of intercalated AO from DNA with the xanthines caffeine (CAF)and aminophylline (AM), utilizing a fluorescence plate reader. FIG. 4-4shows the relationship between the length of time of exposure (no mixingor shaking) and the K_(assoc) value of the xanthine on the removal of AOfrom nuclear chromatin. The example shown in FIG. 4-4 illustrates AOstained (1 mM AQ for 10 sec) chicken erythrocytes smeared slides exposedunder static conditions to aqueous solutions of either 100 mM caffeine(CAF) (n=3), of 100 mM aqueous solution of aminophylline (AM) (n=3), orto distilled water for periods of 10 seconds, 10 minutes, one hour, andtwo hours. Distilled water served as solvent control (n=8). Thereduction of fluorescence (λ_(ex) 485 nm; λ_(em) 530 nm) for AM and CAFfollowed first order decay kinetics with significant fits to theexponential decay model (R² _(CAF)=0.998, P value (runs test)=0.90; R²_(AM)=0.879, P value (runs test)=1.00). A plot of the mean fluorescencevalues percent of control) versus time of exposure to the xanthinecontaining solution shows a good fit to a first order decay modelequation for both CAF (R² _(CAF=)0.998) and AM (R² _(AM=)0.879). Byholding the concentration of both xanthines constant, these resultsindicate that, the removal of AO from nuclear chromatin DNA followsfirst order decay kinetics and is dependent on time of exposure, asexpected by mass action and on the K_(assoc) value of the xanthine.

Lability to acids is a property of DNA. Under mild acid hydrolysis mostof the purine bases are very readily removed from the DNA moleculeresulting in the formation of a potential aldehyde group in the siteswhere the purine bases were bound. This liberated potential aldehydegroup should react with aldehyde reagent. By using the depurinationportion of the Feulgen method to selectively remove the purine bases,the active sites on the DNA polymer available for AO intercalation wereremoved. The phosphate backbone as well as the pyrimidine bases are leftintact and are still able to facilitate electrostatic bonding of the AOmolecule to the DNA.

EXAMPLE 9

In another implementation of this invention, by increasing the bindingaffinity of a modified xanthine with a DNA intercalator, the modifiedxanthine can be used as an interceptor of DNA intercalators. The use ofmodified xanthines is not limited to unbound intercalators, but may beapplied to intercalators bound to polynucleotides as well. The degree ofintercalator removal is related to the ratio of xanthine to theintercalator molecules.

The effect of increasing exposure time and of increasing relativexanthine concentration (molecular ratio of xanthine to AO) on theability of xanthines with increasing K_(assoc) values to block theintercalation of AO into chicken erythrocyte nuclear chromatin,demonstrates that the blocking of AO intercalation into nuclearchromatin, can be controlled, by increasing the molar ratio of xanthineto AO, and further demonstrates that, the xanthines with higherK_(assoc) values require lower molar ratios (xanthine to AO) to have ablocking effect than those with lower K_(assoc) values. The blockingeffect of the relative concentration between AO and the xanthines (molarratio) and their respective K_(assoc) values can be offset by exposuretime to dsDNA. This was expected based on the high K_(assoc) value ofdsDNA with AO (˜4000 M⁻¹, Lyles and Cameron, 2000).

By keeping the exposure times and the molar ratio constant, the removalof AO staining of the nuclear chromatin was directly related to theK_(assoc) values of the xanthines. The value of 256 M⁻¹ for CAF being ata transition K_(assoc) value under the experimental conditions listed inTable 4-3. The ability of the nuclear chromatin to be re-stained furtherindicates that the bonding of AO to dsDNA is in dynamic equilibrium andthat intercalation of AO into nuclear DNA is reversible.

The quantitative results presented in FIG. 4-4 confirmed theobservations with fluorescence microscopy. By holding the molar ratio(concentration) constant, the effect of exposure time was assessed on aquantitative basis. The removal of AO from its intercalation site withinnuclear chromatin significantly fit an exponential decay model with anexcellent fit with more rapid removal of AO from the nuclear chromatinwith AM (K_(assoc) 596 M⁻¹) than with CAF (K_(assoc) 256 M⁻¹).

These experiments with Drosophila salivary gland polytene chromosomesindicate that the bonding of known PAH-DNA intercalators intochromosomal DNA is reversible. The ability of CAF and AM to remove thedifferent PAHs tested demonstrates that CAF and AM can remove PAHintercalators from dsDNA in nuclear chromatin. Xanthines withK_(assoc)>150 M⁻¹ for AO could intercept the AO molecule in aconcentration dependent manner resulting in either the inhibition ofintercalation into nuclear chromatin or the removal of AO from nuclearchromatin once intercalated. Bonding of known intercalators tochromosome DNA is reversible. Efficacy of AO removal from chickenerythrocyte nuclear chromatin and Drosophila polytene chromosome nuclearchromatin was found to be dependent on: 1) the concentration of thexanthine, 2) the concentration of AO, 3) the K_(assoc) value for theAO-xanthine complex, and 4) the time of exposure to the xanthine.

The results also support a general role of specific methyl substitutedxanthines with relatively high K_(assoc) values for DNA intercalators asblockers and scavengers of planar PAH-DNA intercalators from theirbinding sites in chromatin. Thus selected methyl xanthines may serve asdesmutagens. The results also suggest a possible role for interceptormolecules such as CAF in the field of pharmacodynamics, drug delivery,and the lessening of cytotoxic effects caused by certain planarpolyaromatic carcinogens and anti-neoplastic drugs.

The interactions of AO with itself and with DNA are probablynon-covalent. Accordingly, the xanthine, AO, and dsDNA, are in dynamicequilibrium and, therefore, comply with the law of mass action. Based onthis, many polyaromatic mutagens or carcinogens that bind to dsDNAthrough intercalation may be able to bind with a specific xanthine andthus can be blocked from intercalation into dsDNA or removed onceintercalated into dsDNA.

The application of this invention is not limited to the modifiedxanthines tested or to the concentrations used. This invention isapplicable to a wide range of modified xanthines, including, but limitedto 1-methyl xanthine, 1,3-dimethyl xanthine, 3,7-dimethyl xanthine,1,7-dimethyl xanthine, 1,3,7-tri-methyl xanthine, 1,3-dimethyl xanthine,1,3-dimethyl-8-oxy xanthine, 1,3-dimethyl-8-chloro xanthine, and1,3,7,9-tetramethyl-8-oxy xanthine. Additionally, the excess amount ofxanthines need not be limited to that given in Table 4-2. Variations ofthe ratio of xanthine to intercalator are included within the scope ofthis invention. In addition, an polynucleotide, whether it is DNA, RNA,or PNA may be used in either a single, double, or higher order strandedstructure. Furthermore, the use of the interceptor molecule is notlimited to modified xanthines. Any molecule that binds with the DNAintercalator through the law of mass action can be used. Examplesinclude, but are not limited to, purines, pyrimidines, uric acid,porphyrin, polynucleotides, DNA, RNA, PNA, and combinations thereof. Inaddition, this invention also includes the varying the saltconcentration as discussed previously to stabilize the dimer fomation ofintercalator molecules.

EXAMPLE 10

The invention is applicable to to targeting other DNA intercalators. Forexample, the CAF-AO model could be used to design planar interceptormolecules to complex with specific DNA intercalators. An example wouldbe to design an interceptor molecule to complex with planar aflatoxinmolecules, a class of potent DNA intercalator carcinogens thatcontaminate many seeds and grains. Aflatoxins are known to intercalateinto dsDNA at specific sites and are a leading cause of liver cancer inthird world countries. The ability to selectively complex thesemolecules, either as a wash to remove the molecules from foodstuffs oras a treatment for accidental ingestion, could significantly reducetheir hazardous impact. Thus, another implementation of the invention isto remove intercalating molecules that enter into a patient; entry couldbe through mouth, nose, eye, ear, skin, lung, or anus of a patient.

According to one embodiment of the invention, xanthines or othercomplexing agents may be used to remove aflatoxins from foodstuffs. Anaqueous solution of a methyl substituted xanthine containing by weight3% of the complexing agent can be applied to seeds and grainscontaminated with aflatoxins from the fungus Aspergillis niger, suchthat the exposure to the complexing agent significantly reduces theamount of aflatoxin contamination rendering the seed or grain non-toxic.The method is not limited to aflatoxins, but may be applied to,including but not limited to nucleotides, porphorins, xanthines,purines, uric acids, alone or together, as monomers or polymers.

EXAMPLE 11

The cytotoxic side affects of doxorubicin as well as otheranti-neoplastic medicinals are well documented. The design and use of aninterceptor molecule to remove doxorubicin from the blood stream andfrom non-neoplastic cells could be of benefit to patients suffering fromextreme side affects from the repeated use of doxorubicin and otheranti-cancer drugs. Additionally, continued exposure to doxorubicincauses myocardial toxicity. Therefore, the ability to remove doxorubicinfrom myocardial muscle cells through complexation with an interceptormolecule could greatly decrease this hazardous side effect. The abilityto remove or reduce intercellular concentrations of these drugsfollowing successful anti-cancer therapy may significantly reduce thecontinued toxicity to other non-neoplastic cells.

An embodiment shown in FIG. 5-2 illustrates that caffeine binds withdoxorubicin. FIG. 5-2 illustrates the absorbance binding spectra of 10μM doxorubicin titrated with increasing concentration of caffeine.Accordingly caffeine binds with doxorubin. In another implementation,doxorubicin can be bound to chlorophyllin as shown in FIG. 5-3. Theselected spectra in FIG. 5-3 denote important transitions within thetitration series. Prevalent peaks occur at λ_(abs) of 475 nm and 500 nm.These peaks correspond to the prevalent absorbance peaks found atconcentrations of doxorubicin at 10 μM and higher concentrations. Theabsorbance denotes a definitive red-shift in absorbance indicatingcomplexation occurring. The invention is not limited to caffeine or,chlorophyllin, but includes, and is not limited to, other xanthines andporphyrins.

EXAMPLE 12

The use of selected xanthines as carrier molecules, to not only increasethe aqueous solubility of many anti-neoplastic medicinals but also as afacilitator for increased intracellular concentrations of this molecule,is a novel idea. A good example of a potential anti-neoplastic drug thatcould benefit from such a carrier would be paxitaxol, which has a verylow solubility in water and, therefore, tends to attach to plastictubing and to self-precipitate out of solution. The complexation of aninterceptor molecule with this planar drug would change the aqueoussolubility of the drug as well as its transport kinetics. Theoretically,this would increase the intercellular concentrations of the drug whilehaving no interference with the anti-cancer drug's ability to bind tothe DNA within the cell. This is due to the fact that, the carriermolecule-drug complex is in dynamic equilibrium and, therefore, the drugwould be available for intercalation into the DNA based on thedifference in K_(ασσ∘χ) values between the drug and the carrier moleculeand the drug and DNA.

This delivery system can be used for topical, oral, interperitoneal, orintravenous delivery of bioactive agents, specifically, anti-neoplasticagents, to tissues, eukaryotic cells, or prokaryotic cells, or yeasts,or fugi. The carrier may consist of a polymer of purines and/orpyrimidines, and/or xanthines and/or uric acids singularly or incombination with modifications of all or one. The carrier can be singlestranded, double stranded or can contain multiple strands, loops,cross-links or other such modifications that may alter the secondary,tertiary, quaternary structure of the carrier. The carrier may containR-group substitutions around the purine, pyrimidine, xanthine, or uricacid base structure. R-group substitutions may be rendered at the N-1,N-3, N-7, N-9 positions and may involve electron donating, electronwithdrawing, electronegative, electropositive, hydrophilic, hydrophobic,organic or inorganic chemical groups. Additionally, substitutions canoccur at the C-2, C-4, or C-8 positions.

In another example, monomers or polymers of purines, pyrimidines,xanthines, or uric acid may be used for delivery of insoluble bioactiveagents to cells or solubilization of bioactive agents using DNA orDNA-like compounds.

Antineoplastic drugs are known to be useful for the treatment of variouscancers. A typical property of antineoplastic drugs is their lowsolubility in water. This property leads to difficulties when creatingformulations suitable for administration to humans or other animalpatients. Commonly, low concentrations, detergents, oils, liposomes, orother means of solubilization are used.

Taxol (paclitaxel) is supplied by Bristol-Myers Squibb Company as amixture in Cremophor EL (polyethylated castor oil) and dehydratedalcohol. For every 6 mg of paclitaxel, 527 mg of oil and 49.7% alcoholis used. Vials are supplied as 30 mg in 5 mL, 100 mg in 16.7 mL, and 300mg in 50 mL liquid (all 6 mg/mL).

Navelbine (vinorelbine tartrate) is used as a single agent or incombination with cisplatin for the first-line treatment of ambulatorypatients with unresectable, advanced non-small cell lung cancer. It issupplied by Glaxo Wellcome at 10 mg/mL in water without preservatives oradditives.

Ideally, a method for the solubilization of antineoplastic drugs wouldbe available which results in high solubility without the introductionof exogenous, non-native materials to the patient. Thus, there exists aneed for novel administration methods for antineoplastic drugs.

Nucleic acids have been found to be an effective carrier agent for thedelivery of antiheoplastic agents to patients. The nucleicacid-antineoplastic agent composition improves the solubility andclinical application of typically water insoluble drugs.

Nucleic acids have been found to be an effective carrier agent to aid inthe delivery of antineoplastic agents to a patient. A composition isformed comprising one or more antineoplastic agents and nucleic acids.The composition is then administered to the patient. As the nucleicacids are degraded in the patient, the antineoplastic agent becomes moreavailable.

Generally, any antineoplastic agent may be used in connection with thisinvention. Particularly attractive are antineoplastic agents which bindto nucleic acids, and which have low or negligible solubility in water.In such a situation, the nucleic acids can serve as solubilzing agents.The resulting compositions can have a higher concentration ofantineoplastic agents than would a similar composition lacking nucleicacids. Examples of antineoplastic agents include TAXOL (paclitaxel,TAXOL is a registered trademark of Bristol-Myers Squibb Company), 5-FU(5-fluorouracil), ADRIAMYCIN (doxorubicin hydrochloride, ADRIAMYCIN is aregistered trademark of Pharmacia & Upjohn), Cytoxan (cyclophosphamide),Mexate (methotrexate), NOVANTRONE (mitoxantrone, NOVANTRONE is aregistered trademark of Immunex Corporation), NAVELBINE (vinorelbinetartrate, NAVELBINE is a registered trademark of), and TAXOTERE(docetaxel, TAXOTERE is a registered trademark of AventisPharmaceuticals). Additional chemotherapy drugs include Actinomycin D,Altretamine, Asparaginase, Bleomycin, Busulphan, Capecitabine,Carboplatin, Carmustine, Chlorambucil, Cisplatin, Cytarabine,Dacarbazine, Daunorubicin, Doxorubicin, Epirubicin, Etoposide,Fludarabine, Gemeitabine, Hydroxyurea, Idarubicin, Ifosfamide,Irinotecan, Liposomal Doxorubicin, Lomustine, Melphalan, Mercaptopurine,Mitomycin, Oxaliplatin, Procarbazine, Streptozocin, Tamozolomide,Thioguanine, Thiotepa, Tomudex, Topotecan, Treosulfan, Vinblastine,Vincristine, Vindesine. and Vinorelbine. Although most of thesecompounds are soluble in water to some extent, the addition of DNAsignificantly enhances the solubility and structural stability of thebioactive agent or rug whether bound via intercalation or electrostaticattraction or both.

While described in the context of antineoplastic agents, nucleic acidscan be used to improve the solubility of any low water solubility drugwhich binds to or complexes with nucleic acids.

The nucleic acids can generally be any nucleic acid. The nucleic acidcan be DNA, RNA, PNA, or other synthetic nucleic acids. Preferably, thenucleic acids are DNA. The nucleic acids can be single or doublestranded, and preferably are double stranded. The nucleic acids cangenerally be of any size. The nucleic acids can be at least about 4 bp(or bases), 8 bp, 10 bp, 12 bp, 14 bp, 16 bp, 18 bp, 20 bp, 30 bp, 50bp, 100 bp, 200 bp, 500 bp, 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 6 kb, 7 kb, 8kb, 9 kb, 10 kb, 20 kb, 30 kb, 40 kb, 50 kb, 1 mb, or multiple mb.Longer nucleic acids may be desired for their increased stability, whileshorter nucleic acids may be desired for their ease of preparation anddegradation in the patient. The nucleic acids may be chemicallymodified. The nucleic acids may be a mixture of an array of sizes, e.g.nucleic acids isolated from a natural source such as salmon testes orcalf thymus.

The composition may comprise other materials such as buffers,flavorings, sugars, drugs, glycerol, stabilizers, antioxidants, or anyother pharmaceutically acceptable materials.

The solubility of the antineoplastic agent in the inventive compositionis preferably at least about 2 times, 3 times, 4 times, 5 times, 6times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 40times, 50 times, 100 times, 200 times, 300 times, 400 times, 500 times,or 1000 times that in the same composition lacking the nucleic acid.

The patient may be a mammal, bird, fish, or amphibian. The mammal can bea human, dog, cat, rabbit, hamster, ferret, horse, cow, pig, or goatPreferably, the mammal is a human.

The antineoplastic agent—nucleic acid composition can be administered tothe patient in any pharmaceutically or medically acceptable method. Forexample, the composition can be delivered orally, topically,transdermally, by IV, by inhalation, by IP injection, by IM injection,by electroporation, or by liposomes.

As an example, the anti-neoplastic agent Doxorubicin or pasitaxol isdissolved in a primarily aqueous I.V. solution containing anoligonucleotides consisting of 10 mers with 1:1 molar ratio or morespecifically an amount equal to or exceeding 50% of the binding curve;whereas the oligonucleotides is poly adenosine-thymidine in a doublehelix.

The law of mass action can be exploited to determine the requiredconcentrations of interceptor or carrier compound according to thefollowing equations:[X]=K _(assoc) [Y]/(1+K _(assoc) [Y])   Equation I:OR[Z]=K _(assoc) [X]/(1+K _(assoc) [X])*100.   Equation II:

Here K_(assoc) is the binding affinity of the interceptor or complexingagent for the compound or bioactive agent to be complexed; [X] is xtheconcentration or amount of active sites of the interceptor or complexingagent; [Y] is the concentration or amount of active sites of the agentto be complexed; and [Z] is the percent of the binding curve from nobinding to infinite or complete binding. Preferably [Z] should have avalue between 0.1 and 99, and more preferably [Z] should be between 40and 99, and even more preferrably [Z] should be equal to or greater than50. Ideally, the concentration of the complexing or binding agent shouldapproach saturation of the binding curve to the greatest extent possibleconsidering the solubility of the binding agent and the toxicity of saidagent.

In another embodiment of the invention other molecules can be used as acarrier. For example xanthines and chlorophyllin have been shown toexhibit a binding affinity for doxoribucin as illustrated in Example 12.

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

EXAMPLE 13

The the use of specialized xanthines to complex with these PAHs prior tohuman exposure, offers benefits as a preventive modality. For example,the potential hazardous effects from environmental exposure to PAHs,such as benzo(a)pyrene and dimethyl benzanthracene, could besignificantly decreased if the PAHs were first exposed to a xanthinewith a high K_(assoc) value for that particular PAH. For example,professional chimney sweeps, who are occupationally exposed to a varietyof carcinogen PAHs, would be able to significantly reduce their risk ofexposure to these carcinogens. The addition of selected xanthines to thewalls of the chimney prior to cleaning could reduce the biologicalhazards from exposure to these PAHs. Additionally, many occupations thatrisk exposure to planar carcinogens could benefit for either apreventive treatment prior to exposure or a therapeutic treatmentfollowing accidental exposure.

In one embodiment a solution containing an effective amount ofcomplexing or binding agent or agents may be applied to the skin of aperson. The solution may be in a liquid or a gel. For example, a 3%solution of dsDNA would be applied to the skin of chimney sweepers toprevent exposure to bioactive agents and carcinogens; or is appliedpost-exposure as a treatment for the prevention of skin cancer.

In one implementation a modified xanthine or porphyrin may be used as aninterceptor of PAHs. The interceptor need only be delivered to DNAregion that has bound with the PAH in the patient Once the interceptoris delivered to the intercalator molecule in a molar excess, the law ofmass action will drive the reaction such that the binding of theintercalator to the DNA will be reduced.

EXAMPLE 14

A cigarette filtering material consisting of a DNA coating would be agood vehicle to remove PAHs in both primary and secondary smoke. Acigarette filter containing an active DNA surface could extract not onlypolyaromatic hydrocarbons, but also other toxic compounds known tointeract with DNA. In one implementation of this invention, a cigarettefilter would be comprised of dsDNA material. As the smoke passes throughthe DNA in the filter, the PAHs, among others, would bind to the DNA.

EXAMPLE 15

Another implementation of this invention is a method for removing boundintercalated dyes from DNA. Many intercalators, such as AO, have afluorescent tag that can be used for detecting the intercalator bound tothe DNA. The addition of one or more specific dyes or compounds to thecompound of choice (e.g. RNA, DNA, dsDNA, or similar molecules) that hasa specific bining affinity K_(assoc) for that substrate. Once bound tothe substrate, a solution containing one or more compounds with a knownbinding affinity or K_(assoc) for the dye is added to the substrate ineither static or dynamic suspension, washing, rinsing, or other meanswhereby the binding compound can remove, extract, or mask the dye orcompound in a manner following the law of mass action and relating tothe K_(assoc) between the extracting compound and the compound to beextracted, the molar ratios between general and specific bindingaffinities and/or K_(assoc) ranges may be determined and specific and/orgeneral eterminations may be made concerning active sites, geneticmakeup, chemical composition, secondary, tertiary, and quaternarystructure as well as a variety of other issues.

The interceptor molecule may be chosen from, among others, purines,xanthines, pyrimidines, uric acid, porphyrins, and polynucleotides, andpolymers and mixtures thereof. The polynucleotides are preferably doublestranded DNA. The concentration of DNA in the medium should be between0.001% and 10% (wt/wt).

EXAMPLE 16

Another embodiment of the invention is a screening or diagnostic device.In diagnostic or screening studies the interceptor or complexingmolecules may be chemically or physically placed in a gel or other solidstate form so as to immobilize them to preferentially extractbiologically active compounds with significant binding affinities suchthat the flow of either solutions or gases containing the bioactiveagent or agents of choice can be concentrated or extracted from saidmedium.

Additionally, once complexed in a stationary or static form, selectedbioactive agents can be preferentially extracted based on bindingaffinity by further exposure to solutions or gases containing aneffective amount of complexing agent or agents to remove said bioactiveagents from the static or solid state system in aK_(assoc)—concentration dependent manner.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the methods described herein without departing from theconcept, spirit and scope of the invention. More specifically, it willbe apparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention.

1. A method of inhibiting complexation of intercalating molecules withpolynucleotides, comprising: contacting xanthine molecules withintercalating molecules, said xanthine molecule comprising at least oneR-group at the N1, N3, N7, and/or N9 positions.
 2. The method of claim1, wherein the R-group is a methyl group.
 3. The method of claim 1,wherein the xanthine molecule further comprises an oxygen or a halogenat the C8 position, said halogen is selected from the group consistingof Cl, Br, F, and I.
 4. The method of claim 1, wherein the intercalatingmolecule is selected from the group consisting of heterocycliccompounds, said heterocyclic compound selected from the group consistingof acridine orange, doxorubicin, doxorubicin hydrochloride, novantrone,elipticine, ethidium bromide, Hoeschst 33258, and aflatoxin:
 5. Themethod of claim 1, wherein the intercalating molecule is a planarpolyaromatic hydrocarbon molecule.
 6. The method of claim 4, wherein theplanar polyaromatic hydrocarbon molecule is selected from the groupconsisting of benzo(a)pyrene and dimethyl benzathracene.
 7. The methodof claim 1, wherein the the xanthine molecules are in an aqueous medium,concentration of said xanthine solution is in the range from about 1 μuMto about 1M.
 8. The method of claim 1, wherein the xanthine moleculesare in an aqueous medium, concentration of said xanthine solution is inthe range from about 1 uM to about 0.1M.
 9. The method of claim 1,wherein xanthine further comprises an aqueous medium, concentration ofsaid xanthine is in the range from about 100 uM to about 1000 mM. 10.The method of claim 1, wherein the number of R group substitutions onthe xanthine molecule is 5 or less.
 11. The method of claim 1, whereinthe xanthine molecule is selected from a group consisting of 1-methylxanthine, 1,3-dimethyl xanthine, 3,7-dimethyl xanthine, 1,7-dimethylxanthine, 1,3,7-trimethyl xanthine, 1,3-dimethyl xanthine,1,3-dimethyl-8-oxy xanthine, 1,3-dimethyl-8-chloro xanthine,1,3,7,9-tetramethyl-8-oxy xanthine, and mixtures thereof.
 12. The methodof claim 1, wherein the binding affinity between the xanthine moleculeand the intercalating molecule is at least 150 M⁻¹.
 13. The method ofclaim 1, wherein the polynucleotide is selected from a group consistingof DNA, RNA, and PNA.
 14. The method of claim 13, wherein thepolynucleotide is single stranded or double stranded.
 15. The method ofclaim 1, wherein the concentration of xanthine is equal to or greaterthan the concentration of the intercalating molecule
 16. A method ofinhibiting complexation of intercalating molecules with polynucleotides,comprising: contacting interceptor molecules with intercalatingmolecules, said interceptor molecule selected from the group consistingof purines, pyrimidines, xanthines, porphyrins, and polynucleotides. 17.The method of claim 16, wherein the concentration of xanthine is equalto or greater than the concentration of the intercalating molecule
 18. Amethod of removing an intercalating molecule from a polynucleotidemolecule comprising: contacting xanthine molecules with intercalatingmolecules to form a xanthine-intercalating molecule complex and apolynucleotide molecule, said xanthine molecule comprises at least one Rgroup at the N1, N3, N7, and/or N9 positions.
 19. The method of claim18, wherein the R-group is a methyl group.
 20. The method of claim 18,wherein the xanthine molecule further comprises an oxygen or a halogenat the C8 position, said halogen is selected from the group consistingof Cl, Br, F, and I.
 21. The method of claim 18, wherein theintercalating molecule is selected from the group of heterocycliccompounds consisting of acridine orange, doxorubicin, doxorubicinhydrochloride, novantrone, elipticine, ethidium bromide, Hoeschst 33258,and aflatoxin.
 22. The method of claim 18, wherein the intercalatingmolecule is a planar polyaromatic hydrocarbon molecule.
 23. The methodof claim 22, wherein the planar polyaromatic hydrocarbon molecule isselected from the group consisting of benzo(a)pyrene and dimethylbenzathracene.
 24. The method of claim 18, wherein the xanthinemolecules are in an aqueous medium, concentration of said xanthinesolution is in the range from about 1 uM to about 1M.
 25. The method ofclaim 18, wherein the xanthine molecules are in an aqueous medium,concentration of said xanthine solution is in the range from about 1 uMto about 0.1M.
 26. The method of claim 18, wherein the xanthinemolecules are in an aqueous medium, concentration of said xanthine is inthe range from about 100 uM to about 1000 mM.
 27. The method of claim18, wherein the number of R group substitutions on the xanthine moleculeis 5 or less.
 28. The method of claim 18, wherein the xanthine moleculeis selected from a group consisting of 1-methyl xanthine, 1,3-dimethylxanthine, 3,7-dimethyl xanthine, 1,7-dimethyl xanthine, 1,3,7-trimethylxanthine, 1,3-dimethyl xanthine, 1,3-dimethyl-8-oxy xanthine,1,3-dimethyl-8-chloro xanthine, 1,3,7,9-tetramethyl-8-oxy xanthine, andmixtures thereof.
 29. The method of claim 18, wherein the bindingaffinity between the xanthine molecule and the intercalating molecule isat least 150 M⁻¹.
 30. The method of claim 18, wherein the polynucleotideis selected from a group consisting of DNA, RNA, and PNA.
 31. The methodof claim 13, wherein the polynucleotide is single stranded or doublestranded.
 32. The method of claim 18, wherein the concentration of thexanthine molecules is equal to or greater than the concentration of theintercalating molecules.
 33. A composition for efficient delivery of ananti-neoplastic agent to a patient, the composition manufactured from:an antinoeoplastic agent, and a carrier molecule comprising one or moremonomers selected from the group consisting of purines, pyrimidines,xanthines, uric acid, polynucleotides, and mixtures thereof.
 34. Thecomposition of claim 33, wherein the carrier molecule comprises DNA,RNA, or PNA.
 35. The composition of claim 33, wherein the carriermolecule is single stranded, double stranded, partially singledstranded, partially double stranded, multiple stranded, looped, orcross-linked.
 36. The composition of claim 33, wherein the carriermolecule contains an R group substitution.
 37. The composition of claim33, wherein carrier molecule is a xanthine with a substituted R-group atthe N1, N3, N7, or N9 positions.
 38. The composition of claim 33,wherein the carrier molecule contains a substitution that is selectedfrom the group consisting of electronegative, electropositive,hydrophobic, and hydrophilic R-groups.
 39. The composition of claim 38,wherein the substitution on the carrier molecule occurs at the C2, C4,or C8 position.
 40. The composition of claim 33, wherein theantineoplastic agent is selected from the group consisting ofpaclitaxel, 5-fluorouracil, doxorubicin, doxorubicin hydrochloride,cyclophosphamide, methotrexate, mitoxantrone, taxotere, and mixturesthereof.
 41. A composition for efficient delivery of an anti-neoplasticagent to a patient, the composition comprising: an antinoeoplasticagent, and a carrier molecule comprising one or more monomers selectedfrom the group consisting of purines, pyrimidines, xanthines, uric acid,and mixtures thereof.
 42. The composition of claim 41, wherein thecarrier molecule comprises DNA, RNA, or PNA.
 43. The composition ofclaim 41, wherein the carrier molecule is single stranded, doublestranded, partially single stranded, partially double stranded, multiplestranded, looped, or cross-linked.
 44. The composition of claim 41,wherein the carrier molecule contains R-group substitutions around thepurine, pyrimidine, xanthine, or uric acid.
 45. The composition of claim44, wherein the R-group substitution occurs at the N1, N3, N7, or N9position.
 46. The composition of claim 44, wherein the substitution isselected from the group consisting of electronegative, electropositive,hydrophobic, and hydrophilic R-group substitution occurs at the C2, C4,or C8 position.
 47. The composition of claim 41, wherein theantineoplastic agent is selected from the group consisting ofpaclitaxel, 5-fluorouracil, doxorubicin, doxorubicin hydrochloride,cyclophosphamide, methotrexate, mitoxantrone, and taxotere.
 48. A methodfor the efficient delivery of an antineoplastic agent to a patient, themethod comprising: obtaining a mixture comprising an antineoplasticagent and a carrier molecule, said carrier molecule comprising one ormore monomers selected from the group consisting of purines,pyrimidines, xanthines, uric acid, and mixtures thereof; and deliveringthe composition to the patient.
 49. A method for the delivery of anantineoplastic agent to a patient, the method comprising: obtaining amixture comprising an antineoplastic agent and a polynucleotide; anddelivering the composition to the patient.
 50. The method of claim 49,wherein the antinoeplastic agent is selected from the group consistingof paclitaxel, 5-fluorouracil, doxorubicin, doxorubicin hydrochloride,cyclophosphamide, methotrexate, mitoxantrone, and taxotere.
 51. Themethod of claim 49, wherein the patient is a mammal, bird, fish,amphibian, yeast, bacteria, nematode, fungi, or fruit fly.
 52. Themethod of claim 51, wherein the mammal is a human.
 53. The method ofclaim 52, wherein the mammal is selected from the group consisting of amouse, rat, monkey, dog, cat, rabbit, hamster, ferret, horse, cow, pig,and goat.
 54. The method of claim 49, wherein the polynucleotide is DNA,RNA, or PNA.
 55. The method of claim 49, wherein the polynucleotide isdouble stranded or single stranded.
 56. The method of claim 49, whereinthe delivering step comprises oral administration, topicaladministration, transdermal administration, IV administration, IPadministration, IM administration, by electroporation, inhalation, or byliposomes.
 57. A method of removing intercalating carcinogenic moleculesfrom a substance that could be ingested by a mammal comprising:delivering a xanthine molecule to a complex formed by an intercalatingmolecule and a substance capable of entering a body of a mammal; andcomplexing with the intercalating molecule.
 58. The method of claim 57,wherein the intercalating molecule is aflatoxin.
 59. The method of claim57, wherein the substance is coffee.
 60. The method of claim 57, whereinthe complex enters the body through a mouth, nose, eye, ear, skin, lung,or anus of the mammal.
 61. The method of claim 57, wherein the mammal isa human, rodent, rat, mouse, cat, dog, horse, goat, sheep, or any othereukaryote.
 62. A method of removing anti-neoplastic intercalatingcompounds from cellular DNA of a mammal comprising: delivering axanthine molecule to the complex formed by the interaction of anintercalating molecule and cellular DNA and forming axanthine-intercalating molecule complex and cellular DNA.
 63. The methodof claim 62, wherein the intercalating molecule is doxorubicin.
 64. Themethod of claim 62, wherein the mammal is a human, rodent, rat, mouse,cat, dog, horse, goat, or sheep.
 65. A method of reducing theintercellular concentration of an anti-neoplastic intercalatingcompounds, the method comprises delivering a xanthine molecule to theintercellular medium of a mammal.
 66. The method of claim 65, whereinthe intercalating molecule is doxorubicin.
 67. The method of claim 65,wherein the mammal is a human, rodent, rat, mouse, cat, dog, horse,goat, or sheep.
 68. A method of reducing the toxicity to mammals ofpolyaromatic hydrocarbons, the method comprises delivering a xanthinemolecule to the polyaromatic hydrocarbon.
 69. The method of claim 68,where the polyaromatic hydrocarbon is benzo(a)pyrene or dimethylbenzathracene.
 70. The method of claim 72, where the polyaromatichydrocarbon is an aflatoxin.
 71. A method for selectively extractingpolyheterocyclic dyes and compounds from double stranded DNA comprising:contacting interceptor molecules with intercalating molecules forminginterceptor-intercalating molecule complexes and polynucleotides
 72. Themethod of claim 70 wherein the interceptor molecule is selected from thegroup consisting of purine, xanthine, pyrimidine, uric acid, porphorin,nucleic acid and polymers thereof.
 73. A method for selectivelyextracting polyheterocyclic dyes and compounds from double stranded DNAcomprising: contacting multiple types of interceptor molecules withintercalating molecules forming an interceptor-intercalating moleculecomplex and a polynucleotide.
 74. The method of claim 73 wherein theinterceptor molecules are selected from the group consisting of purine,xanthine, pyrimidine, uric acid, porphorin, nucleic acid or polymersthereof.
 75. A method for concentrating a compound in solutioncomprising: complexing a agent with an interceptor molecule.
 76. Themethod of claim 75, wherein the interceptor molecule is selected fromthe group consisting of pyrimidines, purines, polynucleotides,xanthines, uric acid, and porphryins.
 77. The method of claim 75,wherein the interceptor molecule is a polymer comprised of mixtures ofmonomers selected from the group consisting of pyrimidines, purines,polynucleotides, xanthines, uric acid, and porphyrins.
 78. The method ofclaim 75, wherein the agent is in a solid, liquid, or gas phase.
 79. Themethod of claim 75, wherein the agent is selected from the groupconsisting of polyaromatic hydrocarbon, heterocyclic hydrocarbon,carcinogen, mutagen, and teratogen.
 80. A method of concentratingpolyheterocyclic dyes comprising: contacting a polyheterocyclic dyemolecule with an interceptor molecule selected from the group consistingof purines, xanthines, pyrimidines, polynucleotides, uric acid, andporphyrins.
 81. The method of claim 80, wherein the interceptor moleculeis a polymer comprised of mixtures of monomers selected from the groupconsisting of pyrimine, xanthine, uric acid, and porphyrin.
 82. A methodfor extracting polyheterocyclic dyes comprising: contacting doublestranded DNA to polyheterocyclic dyes.
 83. The method of claim 86,wherein the DNA is 1% to 10% (wt/wt).
 83. The method of claim 86,wherein the DNA is 0.1% to 1% (wt/wt).
 85. The method of claim 86,wherein the DNA is 0.001% to 0.1% (wt/wt).